Fatty acids, C6-12, mixed tetraesters with heptanoic acid, pentaerythritol, 3,5,5-trimethylhexanoic acid and valeric acid

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Apr - 31 Jul 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study, tested with the source substance Reaction product of pentaerythritol and trimethylolpropane with n-pentanoic acid, 2-methylbutyric acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (ECHA, 2012)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom, date of inspection: 23 Mar 1998

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/L
- Sampling method: Water samples were taken from the control and the 100 mg/L loading rate WAF test group (replicates R1 - R3 and R4 - R6 pooled) at 0 and 96 h for quantitative analysis. Duplicate samples were taken. A volume of sample was extracted with three portions (3 x 50 mL) of dichloromethane and the extracts filtered through anhydrous sodium sulphate. The combined extracts were evaporated to dryness and the residue re-dissolved in hexane.
- Sample storage conditions before analysis: stored frozen (approximately -20 °C) for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For range-finding test, amounts of test material (210 and 2100 mg) were each separately placed on the surface of 2 L of dechlorinated tap water to give 10 and 100 mg/L loading rates which were then stirred by magnetic stirrer, to achieve a vortex depth of approximately 25 % of the distance to the bottom of the vessel, for 24 hours. The stirring was stopped after 24 h and the mixtures allowed to stand for 1 h prior to removing the aqueous phase or Water Accommodated Fraction by siphon.
Based on the results of the range-finding study a "Limit test" was conducted for the definitive study at a 100 mg/L loading rate WAF to confirm that at the maximum test concentration given in the test guidelines, no effect on algal growth was observed.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: freshwater green algae
- Strain: CCAP 278/4
- Source (laboratory, culture collection): in-house culture after obtained from the Culture Centre for Algae and Protozoa (CCAP), Institute of Freshwater Ecology, Ferry House, Ambleside, Cumbria
- Method of cultivation: The culture was maintained in the laboratory at a temperature of 21 ± 1 °C under continuous illumination (intensity approximately 7000 lux) and constant aeration.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Test temperature:

24 ± 1 °C

pH:

start: 7.5, end: 9.1 - 10.0

Nominal and measured concentrations:

100 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Type: closed
- Material, size: 250 mL glass conical flasks
- Aeration: yes
- Initial cells density: 1.22 x 10^4 cells/mL (mean).
- Control end cells density: 2.82 x 10^6 cells/mL (mean)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes, according to OECD guideline 201


OTHER TEST CONDITIONS
- Adjustment of pH: yes, adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl
- Photoperiod: continuous illumination
- Light intensity and quality: 7000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter Multisizer II Particle Counter

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 10 and 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: no effect on growth at the test concentrations of 10 and 100 mg/L.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

96 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

4 substances available for read across

Adequacy of study:

weight of evidence

Justification for type of information:

see the attached justification in section 13 for the full details.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Reaction product of pentaerythritol and trimethylolpropane with n-pentanoic acid, 2-methylbutyric acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid

Key result

Duration:

96 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Reaction product of pentaerythritol and trimethylolpropane with n-pentanoic acid, 2-methylbutyric acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid

Key result

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Reaction product of pentaerythritol and trimethylolpropane with n-pentanoic acid, 2-methylbutyric acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Hatcol 5326

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Hatcol 5236

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 0.22 - < 0.79 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: Hatcol 3331

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Hatcol 3344

Validity criteria fulfilled:

not applicable

Remarks:

read across

Conclusions:

The read across for substance, CAS: 156558-98-4; EC: 451-190-0; is based upon the analogous substances to which basic form, degree of substitution of functional groups is not considered to effect the proposed read across for the endpoint of toxicity to aquatic algae and cyanobacteria. The NOELR for the substance based on the mean of the information available is deemed to be 25.11 mg/L, to which no adverse effects were noted upto the limit of solubility.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 April 2003 to 18 August 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

During the test duplicate samples for analysis were taken from the test concentration and the blank-control.
Frequency: at t=0 h, t=24 hand 72 h
Volume: 5ml
Storage: Samples were transferred to Analytical Chemistry directly after sampling.

Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae and samples for analysis were taken at the start, after 24 hours and at the end of the test period.

Vehicle:

no

Details on test solutions:

The standard test procedures required generation of test solutions which contained completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that would disturb the test system were prevented as much as possible (e.g. film of the test substance on the water surface).
The batch of HATCOL 3331 tested was a clear colourless liquid with a purity of > 97.3% and the substance was not completely soluble in test medium at the concentration tested. The water solubility of Hatcol3331 at 20.3 ± 0.8°C was determined to be < 2.0x10E-4 g/I, according to the flask method (NOTOX Project 365052). The partition coefficient (n-octanol/water), Pow, was determined to be ≥ 5.1*10E6 (log Pow ≥ 6.7) at 20.3 ± 0.8°C (NOTOX Project 365085).
Preparation of test solutions started with a stock solution of 100 mg/l applying 2 days of magnetic stirring to ensure maximum solubility in test medium. The resulting solution was slightly hazy with a floating layer and test substance droplets. Collection of the water phase by siphoning or centrifugation was not an option considering the specific gravity of the test substance. After the stirring period the mixture was therefore filtered through a paper filter (ca. > 5 µm). The filtrate was very slightly hazy. Note that the medium for the blank-control received the same treatment.
After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, adequate volumes of an algal suspension were added to each replicate providing a cell density of 10E4 cells/l

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Species: Selenastrum capricornutum, strain: NIVA CHL 1.
Source: In-house laboratory culture
Reason for selection: This system is a unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

Hardness (Ca+Mg) 0.24 mmol/I (24 mg CaCO3/l)

Test temperature:

The temperature of the test medium was 23.1°C at the start of the test. During the exposure period the temperature measured in the incubator was maintained between 23.2 and 23.9°C.
Hence, the temperature complied with the requirements as laid down in the protocol (21-25°C, constant within 2°C).

pH:

The pH values measured in the blank-control remained within the limits prescribed by the protocol (6.0-9.0, not varying more than 1.5 unit). However, the pH value measured in the test concentration at the end of the test was slightly higher than the highest limit described in the protocol. This was attributed to a relatively high increase in pH, which was caused by the high algal growth, and therefore not considered to have affected the test.

Dissolved oxygen:

Not specified in the study report.

Salinity:

Not applicable - freshwater study

Nominal and measured concentrations:

Measured test substance concentrations

Details on test conditions:

LIMIT TEST:
TEST CONCENTRATIONS
HATCOL 3331: A 5 µm filtrate prepared at a loading rate of 100 mg/I, the regulatory limit concentration.
Controls: Test medium without test substance or other additives (blank).
Replicates: 6 replicates of the test concentration and the blank control; 2 replicates of the test concentration without algae; 1 extra replicate of the test concentration and the blank-control for sampling purposes.

TEST PROCEDURE AND CONDITIONS
Test type: Static
Test vessels: 100 ml, all-glass
Medium: M2-medium
Cell density: An initial cell density of 1 x 10E4 cells/ml.
Test duration: 72 hours
Illumination: Continuously using TLD-lamps of the type 'Cool-white' of 30 Watt, with a light intensity within the range of 81-111 µE.mE-2.sE-1.
Incubation: During incubation the algal cells were kept in suspension by continuous shaking.

MEASUREMENTS
pH: At the beginning and at the end of the test. The pH of the solutions should preferably not deviate by more than 1.5 units during the test.
Temperature of medium: Continuously in a temperature-control vessel.

RECORDING OF CELL DENSITIES
At the beginning of the test, cells were counted by microscope, using a counting chamber.
Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with immersion probe (pathlength =20 mm). Varian Nederland BV., Houten, The Netherlands. Algal medium was used as blank, and the extra replicate without algae was used as background for the treated solution.
After 24 hours of exposure, the test solutions were so turbid that they disturbed the spectrophotometric measurements. Although algal density was also measured spectrophotometrically after 24 hours, these data were only useful for the blank-controls.
Throughout the remainder of the test, algal density was counted by microscope.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 0.22 - < 0.79 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

dissolved

Basis for effect:

growth rate

Details on results:

Measured test substance concentrations
The test substance consisted of a mixture of molecules with different molecular weights, which differed in water solubility, resulting in a number of peaks in the chromatogram of the test substance solutions. It was not possible to determine which molecule was responsible for the toxicological response, if any. Furthermore, since not all components were quantifiable in the calibration chromatograms, it was not possible to determine the concentration of the total test substance. Therefore, the toxicological evaluation was based on the water-soluble fraction at the loading rate. In addition, the actual concentration was estimated from the three largest peaks (peaks 1, 2 and 3) observed in the chromatograms of HA TCOL 3331.
The analytical results showed that the filtered solution prepared at a loading rate of 100 mg/I contained an initial concentration of 3.3 mg/l (based on peak 1), 4.1 mg/I (based on peak 2) or 4.2 mg/I (based on peak 3). After 24 hours of exposure the test concentration had decreased to below the limit of quantification of 0.2 mg/I (based on peak 1), 0.45 mg/l (based on peak 2) or 0.64 mg/I (based on peak 3). At the end of the test period the test concentration had decreased below the limit of quantification of 0.2 mg/I (based on peaks 1 and 2) or 0.40 mg/I (based on peak 3).
The average exposure concentration, based on peak 1, 2 or 3, respectively, was 0.22, 0.50 and 0.79 mg/l. Hence, average concentrations remained above or approximated the solubility limit of HATCOL 3331 (i.e. < 0.2 mg/I). The observed decrease was probably a consequence of this low solubility.

Mean cell densities
One replicate in the test group was considered to be an outlier, since it only showed a slight increase in cell density, while the other replicates in this group showed maximal growth. In fact, the cell density in the outlier was only 8% of the average value of the other replicates.

Inhibition of cell growth and reduction of growth rate
There was a small but insignificant effect on cell growth among algae exposed to the filtrate (Two-sample t-test, α=0.05). No significant differences were recorded between the values for growth rate in the filtrate (Two-sample t-test, α=0.05).

Experimental conditions
The pH values measured in the blank-control remained within the limits prescribed by the protocol (6.0-9.0, not varying more than 1.5 unit). However, the pH value measured in the test concentration at the end of the test was slightly higher than the highest limit described in the protocol. This was attributed to a relatively high increase in pH, which was caused by the high algal growth, and therefore not considered to have affected the test.
The temperature of the test medium was 23.1 °C at the start of the test. During the exposure period the temperature measured in the incubator was maintained between 23.2 and 23.9°C.
Hence, the temperature complied with the requirements as laid down in the protocol (21-25°C, constant within 2°C).

ACCEPTABILITY OF THE TEST
1. In the blank-control, cell density increased by an average factor of > 16 within 3 days.
2. The test conditions (pH, temperature) were maintained within the limits prescribed by the protocol, except for the pH value measured in the test concentration at the end of the test: this value was slightly higher than the highest limit described in the protocol. This was attributed to a relatively high increase in pH, which was caused by the high algal growth, and therefore not considered to have affected the test.

Results with reference substance (positive control):

The study procedures described in this report were based on the EEC Directive 92/69, Publication No. L383 Part C-3 adopted December, 1992; OECD guideline No. 201, Adopted June 7,1984; and ISO Standard 8692, First edition, 15 November 1989.
This reference test was carried out to check the sensitivity of the test system used by NOTOX to Potassium dichromate (Merck, Art. 4864, Batch K28974764).
Algae were exposed for a period of 72 hours to K2Cr2O7 concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/I and to a blank-control. The initial cell density was 1.0 x 10E4 cells/ml.

Results
Under the conditions of the reference study with Selenastrum capricornutum, potassium dichromate inhibited cell growth of this fresh water algae species at nominal concentrations of 0.56 mg/I and higher and reduced growth rate at 1.0 mg/I and higher.
The EC50 for cell growth inhibition (EBC50: 0-72h) was 0.73 mg/I with a 95 % confidence interval ranging from 0.45 to 1.2 mg/l. The historical ranges of the 72h EC50 for growth inhibition lie between 0.49 and 1.4 mg/l. Hence, the EBC50: 0-72h for the present batch corresponds with this range.
The EC50 for growth rate reduction (ERC50: 0-72h) was 1.2 mg/I with a 95 % confidence interval ranging from 0.90 to 1.6 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/l. Hence, the ERC50: 0-72h for the present batch corresponds with this range.
The protocol, raw data and report of this study are kept in the NOTOX archives. The test described above was performed under GLP conditions with a QA-check.

Reported statistics and error estimates:

Not specified in the study report.

Table 1: Mean cell densities during the limit test

Loading rate HATCOL 3331 (mg/l)	Exposure time (hours)
	0	24	48	72
Blank control	1.0	4.8	37.9	149.2
100	1.0	6.9	31.9	124.6

 

Table 2: Percentage inhibition of cell growth during the limit test

Loading rate HATCOL 3331 (mg/l)	Cell growth (0-72 hrs)
	Mean area (A)	Inhibition (%)
Blank control	2753.60
100	2364.00	14.1

 

Table 3: Percentage reduction of growth rate at different time intervals during the limit test

Loading rate HATCOL 3331 (mg/l)	Mean growth rate
	µ (0-24 hrs)	Reduction (%)	µ (0.48 hrs)	Reduction (%)	µ (0-72 hrs)	Reduction (%)
Blank control	0.06510		0.07567		0.06943
100	0.07976	-22.5	0.071771	5.2	0.06694	3.6

 

Table 4: pH levels recorded during the study

Loading rate HATCOL 3331 (mg/l)	Exposure time (hours)
	0	72
Blank control	8.4	9.0
100	7.8	9.2

 

 

Overview of % inhibition in cell growth and % reduction of growth rate in the reference test:

Concentration K2Cr2O7(mg/l)	Total cell growth:		Growth rate:
	Mean Area 0-72h	Inhibition %	Interval  0-72h	Reduction %
Blank-control	2273.92		0.06422
0.18	2185.2	3.9	0.06409	0.2
0.32	2299.84	-1.1	0.06558	-2.1
0.56	1443.54	36.5	0.05836	9.1
1.0	475.60	79.1	0.03771	41.3
1.8	169.96	92.5	0.01811	71.8
3.2	107.64	95.3	0.01207	81.2

 

Validity criteria fulfilled:

yes

Conclusions:

The NOEC for cell growth inhibition and growth rate reduction was 0.22-0.79 mg/I, obtained in a filtered solution at a loading rate of 100 mg/I the regulatory limit concentration.

Executive summary:

Selenastrum capricornutum, Fresh Water Algal Growth Inhibition Test with HATCOL 3331.

The study procedures described in this report were based on the EEC Directive 92/69, Publication No. L383 Part C-3 adopted December, 1992; OECD guideline No. 201, Adopted June 7,1984; and ISO Standard 8692, First edition, 15 November 1989.

The batch of HATCOL 3331 tested was a clear colourless liquid with a purity of 97.3% and the substance was not completely soluble in test medium at the concentration tested. The waters olubility of Hatcol 3331 at 20.3 ± 0.8°C was determined to be < 2.0x10E-4 g/I, according to the flask method (NOTOX Project 365052). The partition coeffcient (n-octanol/water), Pow, was determined to be ≥ 5.1*10E6 (log Pow ≥ 6.7) at 20.3 ± 0.8°C (NOTOX Project 365085).

Preparation of test solutions started with a stock solution of 100 mg/l applying 2 days of magnetic stirring to ensure maximum solubility in test medium. The resulting solution was slightly hazy with a floating layer and test substance droplets. Collection of the water phase by siphoning or centrifugation was not an option considering the specific gravity of the test substance. After the stirring period the mixture was therefore filtered through a paper filter(ca. > 5 µm). The filtrate was very slightly hazy.

A limit test was performed exposing exponentially growing algal cultures to a filtered (ca. 5µm) HATCOL 3331 solution prepared at a loading rate of 100 mg/I and a blank-control. The initialcell density was 10E4 cells/ml. The total test period was 72 hours. Samples for determination of actual exposure concentrations were taken at the start, after 24 hours of exposure and at theend of the test period.

Analysis of the samples showed that average exposure concentrations were at or above thesolubility limit of HATCOL 3331 (I.e. < 0.2 mg/l).

The study met the acceptability criteria prescribed by the protocol and was considered valid.

HATCOL 3331 induced no inhibition of cell growth or reduction of growth rate in Selenastrum capricornutum at the concentration obtained in a filtered solution prepared at a loading rate of 100 mg/l, corresponding to an average exposure concentration of 0.22-0.79 mg/I (NOEC).

Due to the very low solubility of HATCOL 3331 in water, concentration levels that might be toxic for algae could not be reached.

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 to 17 October 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

During the final test duplicate samples were taken from all concentrations and the blank-control.
Frequency: at t=0 h, t=24 hand t=72 h
Volume: 5ml
Storage: Not applicable, samples were analysed on the day of sampling.
Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae and samples for analysis were taken at the start and the end of the test period.

Vehicle:

no

Details on test solutions:

The standard test procedures required generation of test solutions, which contained completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions.
The testing of concentrations that would disturb the test system were prevented as much as possible (e.g. film of the test substance on the water surface).
HATCOL 3344 is a clear colourless liquid with a purity of 96.9%. The water solubility of HATCOL 3344 at 20.0 ± 0.8°C was determined to be < 2.0x10E-4g/l (pH was 7.4) using the flask method (NOTOX Project 365535).
All test solutions with a loading rate at or above 1.0 mg/l were prepared separately (loading rates of 1.0, 10 and 100 mg/l). These supersaturated solutions were stirred for two days to reach maximum solubility. After the stirring period the 100 mg/l loading rate contained a test substance floating layer and test substance particles, the 10 mg/l loading rate contained test substance particles and the 1.0 mg/l loading rate was observed to be clear and colourless.
Collection of the water phase by siphoning or centrifugation was not an option considering the specific gravity of the test substance (see also NOTOX Project 365535). After the stirring period all three mixtures were therefore filtered through a paper filer (Schleicher and Schuell 604) to remove the larger undissolved test substance particles (ca. > 5µm). Finally a ten-fold dilution was prepared from the filtrate prepared at a loading rate of 1.0 mg/l and indicated as 0.1 mg/l.
All test solutions were clear and colourless.
After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, adequate volumes of an algal suspension were added to each replicate providing a cell density of 10E4 cells/ml.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Species: Selenastrum capricomutum, strain: NIVA CHL 1.
Source: In-house laboratory culture.
Reason for selection: This system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

No specified in the study report.

Test temperature:

The temperature of the test medium was 23.4 °C at the start of the test. During the exposure period the temperature measured in the temperature control vessel was maintained between 23.3 and 24.0 °C, and complied with the requirements as laid down in the protocol (21-25°C, constant within 2°C).

pH:

6.5 – 9.7
pH remained within the limits prescribed by the protocol (pH: 6.0-9.0, not varying by more than 1.5 unit). Exceptions were the blank-control and the lowest test concentration, which had a pH above 9.0 at the end of the test period. Since this deviation could be related to a relatively

Dissolved oxygen:

Not specified in the study report.

Salinity:

Not specified in the study report.

Nominal and measured concentrations:

The concentrations of HATCOL 3344 were determined by Gas Chromatography with mass spectrometric detection (GC-MS). The test substance consisted of a mixture of molecules with different molecular weights, which differed in water solubility, resulting in a number of peaks in the chromatogram of the test substance solutions. It was not possible to determine which molecule was responsible for the toxicological response, if any. Furthermore, since not all components were quantifiable in the calibration chromatograms, it was not possible to determine the concentration of the total test substance. Therefore, the toxicological evaluation was based on the water-soluble fraction at the loading rate. In addition, the actual concentration was estimated from the two largest peaks (peaks 1 and 2) observed in the chromatograms of HATCOL 3344.
The analytical results showed that the filtered solution prepared at a loading rate of 100 mg/l had an initial concentration of 0.65 mg/l (when based on peak 1) or was below the limit of quantification (i.e. below 0.1 mg/l, when based on peak 2). The filtered solution prepared at a loading rate of 10 mg/l had an initial concentration of 0.12 mg/l (when based on peak 1) or was below the limit of quantification (i.e. below 0.1 mg/l, when based on peak 2). After 24 and 72 hours of exposure both test concentrations had decreased below the limit of quantification (based on both peaks). The lower test concentrations were below the limit of quantification from the start of exposure (based on both peaks).
The average exposure concentration at a loading rate of 100 mg/l, based on peak 1, was 0.18 mg/l, while the average exposure concentration at a loading rate of 10 mgl1, based on peak 1, was 0.08 mg/l. Hence, the average exposure concentration at loading rates of 10 and 100 mg/l approximated the solubility limit of HATCOL 3344 (i.e. < 0.2 mg/l. The observed decrease was probably a consequence of this extremely low solubility.

Details on test conditions:

Test concentrations
HATCOL 3344: 5 µm filtered solutions prepared at loading rates of 1.0, 10 and 100 mg/l and a ten-fold dilution of the filtrate prepared at a loading rate of 1.0 mg/l.
Controls: Test medium without test substance or other additives (blank-control).
Replicates: 3 replicates of each test concentration. 6 replicates of the blank-control. 2 replicates of the highest concentration without algae. 1 replicate of the lower test concentrations without algae. 1 extra replicate of each solution for sampling purposes.

Test procedures and conditions
Test duration: 72 hours
Test type: Static
Test vessels: 100 ml, all-glass, containing 50 ml of test medium
Medium: M2
Cell density: An initial cell density of 1 x 10E4 cells/ml.
Illumination: Continuously using TLD-Iamps of the type 'Cool-white' of 30 Watt, with a light intensity within the range of 78 to 111 µE.mE-2.sE-1.
Incubation: During incubation the algal cells were kept in suspension by continuous shaking.

Measurements
pH: At the beginning and at the end of the test. The pH of the solutions should preferably not deviate by more than 1.5 units during the test.
Temperature of medium: Continuously in a temperature control vessel.

Recording of cell densities: At the beginning of the test, cells were counted by microscope, using a counting chamber.
Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with immersion probe (pathlength =20 mm). Varian Nederland BV., Houten, The Netherlands. Algal medium was used as blank. In order to correct for turbidity one control, without algae, was measured for the 100 mg/l loading rate at each time interval and also for the 10 mg/l loading rate at the end of the test period.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Final test
Measured test substance concentrations: The concentrations of HATCOL 3344 were determined by Gas Chromatography with mass spectrometric detection (GC-MS). The test substance consisted of a mixture of molecules with different molecular weights, which differed in water solubility, resulting in a number of peaks in the chromatogram of the test substance solutions. It was not possible to determine which molecule was responsible for the toxicological response, if any. Furthermore, since not all components were quantifiable in the calibration chromatograms, it was not possible to determine the concentration of the total test substance. Therefore, the toxicological evaluation was based on the water-soluble fraction at the loading rate. In addition, the actual concentration was estimated from the two largest peaks (peaks 1 and 2) observed in the chromatograms of HATCOL 3344.
The analytical results showed that the filtered solution prepared at a loading rate of 100 mg/l had an initial concentration of 0.65 mg/l (when based on peak 1) or was below the limit of quantification (i.e. below 0.1 mg/l, when based on peak 2). The fitlered solution prepared at a loading rate of 10 mg/l had an initial concentration of 0.12 mg/l (when based on peak 1) or was below the limit of quantification (i.e. below 0.1 mg/l, when based on peak 2). After 24 and 72 hours of exposure both test concentrations had decreased below the limit of quantification (based on both peaks). The lower test concentrations were below the limit of quantification from the start of exposure (based on both peaks).
The average exposure concentration at a loading rate of 100 mg/l, based on peak 1, was 0.18 mg/l, while the average exposure concentration at a loading rate of 10 mg/l, based on peak 1, was 0.08 mg/l. Hence, the average exposure concentration at loading rates of 10 and 100 mg/l approximated the solubility limit of HATCOL 3344 (i.e. < 0.2 mg/l. The observed decrease was probably a consequence of this extremely low solubility.

Inhibition of cell growth and reduction of growth rate: Inhibition of cell growth was 25% at loading rates of 1.0 and 10 mg/l and increased to 87% at a loading rate of 100 mg/l. No inhibition of cell growth was observed at a loading rate of 0.1 mg/l. Statistically significant inhibition of cell growth was found at loading rates of 1.0 mg/l and higher (Tukey test, α = 0.05).
Growth rates were in the range of the controls at the loading rate of 0.1 mg/l during the 72-hour test period. Reduction of growth rate was ca. 6% at loading rates of 1.0 and 10 mg/l and increased to 43% at a loading rate of 100 mg/l. Statistically significant reduction of growth rate was found at loading rates of 1.0 mg/l and higher (Tukey test, α = 0.05).

Determination of effect concentrations: Since the effects at the filtered solutions prepared at loading rates of 1.0 and 10 mg/l were approximately the same, EC-values were based only on the results at the filtered solutions prepared at loading rates of 10 and 100 mg/l and have to be considered as indicative.

Experimental conditions: pH remained within the limits prescribed by the protocol (pH: 6.0-9.0, not varying by more than 1.5 unit). Exceptions were the blank-control and the lowest test concentration, which had a pH above 9.0 at the end of the test period. Since this deviation could be related to a relatively high rate of algal growth it was not considered to have affected the validity criteria of the test.
Note that the test substance appeared to have an effect on the pH of the M2-medium at the start of the test. However, during exposure pH increased in all vessels, indicating that the initial pH reduction did not affect growth to a large extent.
The temperature of the test medium was 23.4°C at the start of the test. During the exposure period the temperature measured in the temperature control vessel was maintained between 23.3 and 24.0°C, and complied with the requirements as laid down in the protocol (21-25°C, constant within 2°C).

Results with reference substance (positive control):

Selenastrum capriconutum, strain: NIVA CHL 1. Fresh water algal growth inhibition test with potassium dichromate (NOTOX Project 381869).
Algae were exposed for a period of 72 hours to K2Cr2O7(POTASSIUM DICHROMATE) concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l and to a blank-control. The initial cell density was 1.0 x 104 cells/ml.
Results:
Overview of % inhibition in cell growth and % reduction of growth rate In the reference test:
Concentration Total cell growth: Growth rate:
K2Cr2O7 Mean Area Inhibition Interval Reduction
(mg/l) 0-72h % 0-72h %
Blank-control 2342.68 0.06742
0.18 2290.52 2.2 0.06740 0.0
0.32 2068.32 11.7 0.06584 2.4
0.56 1328.36 43.3 0.05953 11.7
1.0 328.48 86.0 0.03535 47.6
1.8 49.20 97.9 0.00000 100.0
3.2 24.08 99.0 0.00000 100.0

Under the conditions of the reference study with Selenastrum capricomutum, potassium dichromate inhibited cell growth and reduced growth rate of this fresh water algae species at nominal concentrations of 0.56 mg/l and higher.
The EC50 for cell growth inhibition (EBC50: 0-72h) was 0.59 mg/l with a 95 % confidence interval ranging from 0.38 to 0.92 mg/l. The ISO-Standard gives a range for the EC50 for cell growth inhibition from 0.20 to 0.75 mg/l based on 16 inter-laboratory tests. Hence, the EBC50: 0-72h for the present batch is within this range.
The EC50 for growth rate reduction (ERC50: 0-72h) was 0.89 mg/l with a 95 % confidence interval ranging from 0.56 to 1.4 mg/l. The ISO-Standard gives a range for the EC50 for cell growth inhibition from 0.60 to 1.03 mg/l based on 16 inter-laboratory tests. Hence, the ERC50: 0-72h for the present batch is within this range.

Reported statistics and error estimates:

Not specified in the study report

Table 1 Mean cell densities (x 10⁴cells/ml) during the final test

Loading rate Hatcol 3344 (mg/l)	Exposure time (hours)
	0	24	48	72
Blank-control	1.0	5.5	41.0	174.7
0.1	1.0	5.6	43.9	171.5
1.0	1.0	5.1	31.1	129.7
10	1.0	5.9	33.0	124.2
100	1.0	2.7	7.8	19.0

 

Table 2 Percentage inhibition of cell growth during the final test

Loading rate Hatcol 3344 (mg/l)	Cell growth (0-72hrs)
	Mean area (A)	Inhibition (%)
Blank-control	3153.98
0.1	3187.60	-1.1
1.0	2364.48	25.0
10	2364.64	25.0
100	420.68	86.7

 

Table 3 Percentage reduction of growth rate at different time intervals during the final test

Loading rate Hatcol 3344 (mg/l)	Mean growth rate
	µ (0-24 hrs)	Reduction (%)	µ (0-48 hrs)	Reduction (%)	µ (0-72 hrs)	Reduction (%)
Blank-control	0.07110		0.07728		0.07168
0.1	0.07197	-1.2	0.07881	-2.0	0.07138	0.4
1.0	0.06772	4.7	0.07159	7.4	0.06752	5.8
10	0.07376	-3.7	0.07283	5.7	0.06697	6.6
100	0.04162	41.5	0.04264	44.8	0.04091	42.9

 

Table 4 Effect parameters

Parameter	Loading rate HATCOL 3344 (mg/l)	95%-confidence Interval
Observed NOELR	0.1
72h EBL10	*	*
72h EBL50	25	19-35
72h ERL10	12	10-15
72h ERL50	>100

*Could not be calculated using log-linear regression

 

Table 5 pH levels recorded during the final test

Loading rate Hatcol 3344 (mg/l)	Exposure time (hours)
	0	72
Blank-control	8.4	9.5
0.1	8.3	9.7
1	8.1	8.8
10	7.8	8.6
100	6.5	7.8

 

Validity criteria fulfilled:

yes

Conclusions:

The EL50 for cell growth inhibition (EBL50: 0-72h) was 25 mg/l with a 95 % confidence interval ranging from 19 to 35 mg/l.
The EL50 for cell growth inhibition (EBL10: 0-72h) could not be determined using log-linear regression.
As growth rate is derived from the slope under the growth curve in a logarithmic plot, the measure of the specific growth rate is preferable over biomass following from the mathematical nature of exponential growth.
The EL50 for growth rate reduction (ERL50: 0-72h) was beyond the range tested.
The EL10 for growth rate reduction (ERL10: 0-72h) was 12 mg/l with a 95 % confidence interval ranging from 10 to 15 mg/l.
The NOELR for both cell growth inhibition and growth rate reduction was 0.1 mg/l.

Executive summary:

Selenastrum capricorutum. Fresh Water Algal Growth Inhibition Test with HATCOL 3344.

The study procedures described in this report were based on the ISO International Standard8692, 1989. In addition, the procedures were designed to meet the test methods and validitycriteria of the EEC directive 92169. Part C.3, 1992 and the OECD guideline No. 201, 1984.

HATCOL 3344 is a clear colourless liquid with a purity of 96.9%. The water solubility of HATCOL 3344 at 20.0 ± 0.8°C was determined to be < 2.0x10e-4 g/l (pH was 7.4) using the flask method (NOTOX Project 365535).

All test solutions with a loading rate at or above 1.0 mg/l were prepared separately (loadingrates of 1.0, 10 and 100 mg/I). These supersaturated solutions were stirred for two days toreach maximum solubility. Collection of the water phase by siphoning or centrifugation was not an option considering the specific gravity of the test substance (see also NOTOX Project 365535). After the stirring period all three mixtures were therefore filtered through a paper filter (Schleicher and Schuell 604, ca. > 5µm). Finally a ten-fold dilution was prepared from the filtrate prepared at a loading rate of 1.0 mg/l. All test solutions were clear and colourless.

A final test was performed exposing exponentially growing algae to 5 µm filtered solutions prepared at loading rates of 1.0, 10 and 100 mg/l and in addition to a ten-fold dilution of the filtrate prepared at a loading rate of 1.0 mg/l for a period of 72 hours. Samples for analytical confirmation of actual exposure concentrations were taken from all test solutions at the start, after 24 hours of exposure and at the end of the test.

Analysis of the samples showed that the average exposure concentration in the WAFs prepared at loading rates of 10 and 100 mg/l approximated the solubility limit of HATCOL 3344 (i.e. < 0.2 mg/l). However, since not all components were quantifiable in the calibration chromatograms, it was not possible to determine the concentration of the total test substance. Therefore, the toxicological evaluation was based on loading rates.

In the controls, cell density increased by an average factor of > 16 within 3 days. Temperature and pH remained within the ranges prescribed by the protocol, except for the pH of the blank-control and the lowest test concentration, which increased above 9.0 at the end of the test period. Since this deviation could be related to a relatively high rate of algal growth it was not considered to have affected the validity criteria of the test.

Due to the fact that test concentrations below or approximating the water solubility level of HATCOL 5236 could not be analysed as these were below the limit of quantification, a different approach was followed for the calculation of EC and NOEC-values.

To this respect the following text quoted from the OECD guidance on classification of chemicals is applicable: 'Many substances covered by the classification scheme are in fact mixtures, for which measurement of exposure concentrations is difficult, and in some cases impossible.

Substances such as petroleum distillate fractions, polymers, substances with significant levels of impurities, etc can pose special problems since the toxic concentration is difficult to define and impossible to verify. Typical testing procedures often rely on the formation of a Water Soluble Fraction WSF) or Water Accommodated Fraction WAF) and data are reported in termsof loading rates. These data may be used in applying the classification criteria.' (Organization for Economic Co-operation and Development (OECO), OECO guidance document on the use of the harmonised system for the classification of chemicals which are hazardous for the aquatic environment, OECD series on testing and assessment number 27, July 23,2001).

HATCOL 3344 reduced growth rate of Selenastrum capricornutum significantly at loading rates of 1.0 mg/l and higher.

The Table below gives the effect parameters based on loading rates.

Parameter	Loading rate HATCOL 3344 (mg/l)	95%-confidence Interval
Observed NOELR	0.1
72h-EBL10	*	*
72h-EBL0	25	19-35
72h ERL10	12	10-15
72h ERL50	>100

*Could not be calculated using log-linear regression.

As growth rate is derived from the slope under the growth curve in a logarithmic plot, the measure of the specific growth rate is preferable over biomass following from the mathematical nature of exponential growth.

Reference 5

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 May 2003 to 13 October 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

During the final test duplicate samples were taken from all concentrations and the blank-control.
Frequency: at t=0 h and t=72 h
Volume: 5ml
Storage: Not applicable, samples were analysed on the day of sampling.
Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae and samples for analysis were taken at the start and the end of the test period.

Vehicle:

no

Details on test solutions:

The standard test procedures required generation of test solutions which contained completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions.
The testing of concentrations that would disturb the test system were prevented as much as possible (e.g. film of the test substance on the water surface).
HATCOL 5236 is a clear pale yellow liquid with a purity of 97.6%. The water solubility of
HATCOL 5236 at 20.0 ± 0.8°C was determined to be < 2.0x10e-4 g/l using the flask method (NOTOX Project 365041 ).
All test solutions with a loading rate at or above 1.0 mg/l were prepared separately. These supersaturated solutions were stirred for two days to reach maximum solubility. After the stirring period the mixtures contained a test substance floating layer and test substance particles.
Collection of the water phase by siphoning or centrifugation was not an option considering the specific gravity of the test substance {see also NOTOX Project 365041 ). After the stirring period the mixtures were therefore filtered through a paper filter (Schleicher and Schuell 604) to remove the larger undissolved test substance particles (ca.> 5 µm). Finally a ten-fold dilution was prepared from the filtrate prepared at a loading rate of 1.0 mg/l and indicated as 0.1 mg/l.
All test solutions were clear and colourless, except for the two highest test concentrations, which were very slight to slightly hazy/cloudy, respectively. Note that the blank-control received the same treatment. After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, adequate volumes of an algal suspension were added to each replicate providing a cell density of 10e4 cells/ml.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Species: Selenastrum caprtcomutum, strain: NIVA CHL 1.
Source: In-house laboratory culture.
Reason for selection: This system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.

Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 23±2°C.
Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

Stock culture medium: M1; formulated using Milli-Ro water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
NaNO3 500 mg/l
K2HPO4 40 mg/l
MgSO4•7H2O 76 mg/l
Na2CO3•10H2O 54 mg/l
C6H8O7•H2O 6 mg/l
NH4NO3 330 mg/l
CaCl2•H2O 36 mg/l
C8H5FeO7•xH2O 6 mg/l
H3BO3 2.9 mg/l
MnCl2•4H2O 1.81 mg/l
ZnCl2 0.11 mg/l
CUSO4•5H2O 0.08 mg/l
(NH4)5Mo7O24•4H2O 0.018 mg/l

Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 2.104 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Pre-culture medium: M2; according to the 180-Standard "Algal growth inhibition test" Nov. 1989; formulated using Milli-Q water (tap water purified by reverse osmosis (milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges: Milli-Q water; Millipore Corp., Bedford, Mass., USA) preventing precipitation and with the following composition:
NH4Cl 15 mg/l
MgCl2•6H2O 12 mg/l
CaCl2•2H2O 18 mg/l
MgSO4•7H2O 15 mg/l
KH2PO4 1.6 mg/l
FeCl3•6H2O 80 µg/l
Na2EDTA•2H2O 100 µg/l
H3BO3 185 µg/l
MnCl2•4H2O 415 µg/l
ZnCl2 3 µg/l
CoCl2•6H2O 1.5 µg/l
CuCl2•2H2O 0.01 µg/l
Na2MoO4•2H2O 7 µg/l
NaHCO3 50 mg/l
Hardness (Ca+Mg) 0.24 mmol/l (254 mg CaCO3/l)
pH 8.3 ± 0.2

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

Hardness (Ca+Mg) 0.24 mmol/l (254 mg CaCO3/l)

Test temperature:

The temperature of the test medium was 23.4°C at the start of the test. During the exposure period the temperature measured in the temperature control vessel was maintained between 22.1 and 22.9°C, and complied with the requirements as laid down in the protocol (21-25°C, constant within 2°C).

pH:

pH: 6.0-9.0, not varying by more than 1 unit.

Dissolved oxygen:

No specified in the study report.

Salinity:

No specified in the study report.

Nominal and measured concentrations:

Measured test substance concentrations
The concentrations of HATCOL 5236 were determined by Gas Chromatography with mass spectrometric detection (GC-MS). The test substance consisted of a mixture of molecules with different molecular weights, which differed in water solubility, resulting in a number of peaks in the chromatogram of the test substance solutions. It was not possible to determine which molecule was responsible for the toxicological response, if any. Furthermore, since not all components were quantifiable in the calibration chromatograms, it was not possible to determine the concentration of the total test substance. Therefore, the toxicological evaluation was based on the water-soluble fraction at the loading rate. In addition, the actual concentration was estimated from the two largest peaks (peaks 1 and 2) observed in the chromatograms of HATCOL 5236.
The analytical results showed that the filtered solution prepared at a loading rate of 100 mg/l had an initial concentration of 1.4 mg/l (when based on peak 1) or 0.34 mg/l (when based on peak 2). After 72 hours of exposure the test concentration had decreased below the limit of quantification (i.e. below 0.1 mg/l, based on both peaks). The lower test concentrations were below the limit of quantification from the start of exposure (based on both peaks).
The average exposure concentration at a loading rate of 100 mg/l, based on peak 1, was 0.27 mg/l, while the average exposure concentration, based on peak 2, was 0.13 mg/l. Hence, the average exposure concentration at a loading rate of 100 mg/l remained above or approximated the solubility limit of HATCOL 5236 (i.e. < 0.2 mg/l). The observed decrease was probably a consequence of this extremely low solubility. Since toxicologically relevant test concentrations could not be analysed, biological results were based on loading rates.

Details on test conditions:

TEST CONCENTRATIONS
HATCOL 5236: 5 µm filtered solutions prepared at loading rates of 1.0, 10 and 100 mg/l and a ten-fold dilution of the filtrate prepared at a loading rate of 1.0 mg/l.
Controls: Test medium without test substance or other additives (blank-control).
Replicates: 3 replicates of each test concentration. 6 replicates of the blank-control. 2 replicates of the highest concentration without algae. 1 replicate of the lower test concentrations without algae.

TEST PROCEDURE AND CONDITIONS
Test type: Static
Test vessels: 100 ml, all-glass
Medium: M2
Cell density: An initial cell density of 1 x 104 cells/ml.
Test duration: 72 hours
Illumination: Continuously using TLD-lamps of the type 'Cool-white' of 30 Watt, with a light Intensity within the range of 76 to 105 µE.m-2.s-1
Incubation: During incubation the algal cells were kept in suspension by continuous shaking.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Inhibition of cell growth and reduction of growth rate: Inhibition of cell growth increased with increasing loading rates of HATCOL 5236 from 1.0 mg/l upwards resulting in 93% inhibition at a loading rate of 100 mg/l. Statistically significant inhibition of cell growth was found at loading rates of 1 a mg/l and higher (Tukey test: a=0.05).
Growth rates of algae exposed to loading rates of 1 0 mg/l and higher were increasingly reduced. Statistically significant reduction of growth rate was found at loading rates of 10 mg/l and higher (Tukey test: 0=0.05).
HATCOL 5236 is a mixture of pentaerythritol-bound fatty acids with each molecule containing four chains of fatty acids with various length. Fatty acids can be absorbed by algae and possibly even be metabolised. At the lowest test concentration algal cells had significantly grown during the test period when compared to the blank-control..
The 'roughly' filtered solutions prepared at loading rates of 10 and 100 mg/l were emulsions of micro-scale droplets. The presence of these droplets could have influenced algal cells causing inhibition of cell growth.

Experimental conditions: pH remained within the limits prescribed by the protocol (pH: 6.0-9.0, not varying by more than 1 unit). Note that the test substance appeared to have an effect on the pH of the M2-medium at the start of the test. However, during exposure this effect was straightened out.
The temperature of the test medium was 23.4°C at the start of the test. During the exposure period the temperature measured in the temperature control vessel was maintained between 22.1 and 22.9°C, and complied with the requirements as laid down in the protocol (21-25°C, constant within 2°C).

The EL50 for total cell growth inhibition (EBL50: 0-72h) was 8.0 mg/l with a 95% confidence interval ranging from 3.5 to 19 mg/l.
The EL10 for total cell growth Inhibition (EBL10: 0-72h) was 0.71 mg/l with a 95% confidence interval ranging from 0.28 to 1.80 mg/l.
As growth rate is derived from the slope under the growth curve in a logarithmic plot, the measure of the specific growth rate is preferable over biomass following from the mathematical nature of exponential growth. In addition total cell growth is depended on growth rate, whereas growth rate is independent from cell density during exponential cell growth as was the case in the present test for all concentrations. For evaluation of the results and for classification of the test substance it is therefore advised to use growth rate rather than total cell growth.
The EL50 for growth rate reduction (ERL50: 0-72h) was 47 mg/l with a 95% confidence interval ranging from 9.9 to 230 mg/l.
The EL10 for growth rate reduction (ERL10: 0-72h) was 2.0 mg/l with a 95% confidence interval ranging from 0.41 to 9.6 mg/l.
The NOELR for both cell growth inhibition and growth rate reduction was 1.0 mg/l.

Results with reference substance (positive control):

This reference test was carried out to check the sensitivity of the test system used by NOTOX to Potassium dichromate (Merck, Art. 4864, Batch K28974764).
Algae were exposed for a period of 72 hours to K2Cr20 7 concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l and to a blank-control. The initial cell density was 1.0 x 104 cells/ml.
Under the conditions of the reference study with Selenastrum capricomutum, potassium dichromate inhibited cell growth of this fresh water algae species at nominal concentrations of 0.56 mg/l and higher and reduced growth rate at 1.0 mg/l and higher.
The EC50 for cell growth inhibition (ERC50: 0-72h) was 0.73 mg/l with a 95% confidence interval ranging from 0.45 to 1.2 mg/l. The historical ranges of the 72h EC50 for growth inhibition lie between 0.49 and 1.4 mg/l. Hence, the ERC50: 0-72h for the present batch corresponds with this range.
The EC50 for growth rate reduction (ERC50: 0-72h) was 1.2 mg/l with a 95% confidence interval ranging from 0.90 to 1.6 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/l. Hence, the ERC50: 0-72h for the present batch corresponds with this range.

Reported statistics and error estimates:

ACCEPTABILITY OF THE TEST
1. In the controls, cell density increased by an average factor of > 16 within 3 days.
2. Further, test conditions (temperature and pH) remained within the ranges prescribed by the protocol.
DETERMINATION OF EL-VALUES
The EL-values with respective 95% confidence intervals have been calculated from growth inhibition and growth rate reduction versus the log of the loading rate curves.

Mean cell densities (x 10⁴cells/ml) during the final test

Loading rate Hatcol 5236 (mg/l)	Exposure time (hours)
	0	24	48	72
Blank-control	1.0	2.7	12.5	45.4
0.10	1.0	3.6	20.8	81.8
1.0	1.0	3.7	10.1	36.0
10	1.0	2.4	7.6	20.2
100	1.0	1.3	1.7	3.9

 

Percentage inhibition of cell growth during the final test

Loading rate Hatcol 5236 (mg/l)	Cell growth (0-72 hrs)
	Mean area (A)	Inhibition (%)
Blank control	849.28
0.10	1507.28	-77.5
1.0	702.80	17.2
10	421.76	50.3
100	59.76	93.0

 

Percentage reduction of growth rate at different time intervals during the final test

Loading rate Hatcol 5236 (mg/l)	Mean growth rate
	µ (0-24 hrs)	Reduction (%)	µ (0-48 hrs)	Reduction (%)	µ (0-72 hrs)	Reduction (%)
Blank control	0.04045		0.05251		0.05285
0.10	0.05360	-32.5	0.06320	-20.3	0.06115	-15.7
1.0	0.05404	-33.6	0.04811	8.4	0.04971	5.9
10	0.03616	10.6	0.04224	19.6	0.04168	21.1
100	0.00980	75.8	0.01125	78.6	0.01903	64.0

 

pH levels recorded during the final study

Loading rate Hatcol 5236 (mg/l)	Exposure time (hours)
	0	72
Blank control	8.2	8.0
0.10	8.2	8.8
1.0	8.1	7.9
10	7.1	7.7
100	6.5	7.4

 

EL-values for growth inhibition

Loading rate	X	Y	Slope:			37.8580
(mg/l)	Log conc. (mg/l)	Inhibition (%)	Intercept:			15.6588
0.10	*	-81.1	Multiple R:			0.9874
0.10	*	84.8	n = number of observations:			9
0.10	*	-66.5
1.0	0.000	7.9	Regression line: Y = 37.86X + 15.56
1.0	0.000	22.8
1.0	0.000	21.0	Prediction of X values based in known Y values
10	1.000	54.4	Known Y Inhibition (%)	10^Xreg(mg/l)	10^X95% -(mg/l)		10^X95%+(mg/l)
10	1.000	50.6
10	1.000	46.0	10	0.71	0.28		1.80
100	2.000	95.0	25	1.76	0.73		4.28
100	2.000	92.4	50	8.07	3.45		18.91
100	2.000	91.5	100	168.98	65.88		433.44

*Not included in the calculations

 

EL-values for growth rate reduction

Loading rate	X	Y	Slope:			29.0289
(mg/l)	Log conc. (mg/l)	Inhibition (%)	Intercept:			1.3333
0.10	*	-16.8	Multiple R:			0.9614
0.10	*	-16.7	n = number of observations:			9
0.10	*	-13.6
1.0	0.000	2.9	Regression line: Y = 29.03X + 1.33
1.0	0.000	8.1
1.0	0.000	6.8	Prediction of X values based in known Y values
10	1.000	23.8	Known Y Inhibition (%)	10^Xreg(mg/l)	10^X95% -(mg/l)		10^X95%+(mg/l)
10	1.000	20.4
10	1.000	19.3	10	1.99	0.41		9.61
100	2.000	65.9	25	6.54	1.42		30.00
100	2.000	63.4	50	47.48	9.87		228.49
100	2.000	62.7

*Not included in the calculations

 

Overview of % inhibition in cell growth and % reduction of growth rate in the reference test

Concentration K2Cr2O7(mg/l)	Total cell growth:		Growth rate:
	Mean area	Inhibition	Interval	Reduction
	0-72h	%	0-72h	%
Blank-control	2273.92		0.06422
0.18	2185.92	3.9	0.06409	0.2
0.32	2299.84	-1.1	0.06558	-2.1
0.56	1443.54	36.5	0.05836	9.1
1.0	475.60	79.1	0.03771	41.3
1.8	169.96	92.5	0.01811	71.8
3.2	107.64	95.3	0.01207	81.2

 

Validity criteria fulfilled:

yes

Conclusions:

The EL50 for total cell growth inhibition (EBL50: 0-72h) was 8.0 mg/l with a 95% confidence interval ranging from 3.5 to 19 mg/l.
The EL10 for total cell growth Inhibition (EBL10: 0-72h) was 0.71 mg/l with a 95% confidence interval ranging from 0.28 to 1.80 mg/l.
The EL50 for growth rate reduction (ERL50: 0-72h) was 47 mg/l with a 95% confidence interval ranging from 9.9 to 230 mg/l.
The EL10 for growth rate reduction (ERL10: 0-72h) was 2.0 mg/l with a 95% confidence interval ranging from 0.41 to 9.6 mg/l.
The NOELR for both cell growth inhibition and growth rate reduction was 1.0 mg/l.

Executive summary:

Selenastrum capricomutum, Fresh Water Algal Growth Inhibition Test with HATCOL 5236.

 

The study procedures described in this report were based on the ISO International Standard 6341, 1996. In addition, the procedures were designed to meet the test methods and validity criteria of the EEC directive 92/69, Part C.3, 1992 and the OECD guideline No. 201 Part I, 1984.

 

HATCOL 5236 is a clear pale yellow liquid with a purity of 97.6%. The water solubility of HATCOL 5236 at 20.0± 0.8°C was determined to be< 2.0x10 ^-4g/l using the flask method (NOTOX Project 365041).

 

All test solutions with a loading rate at or above 1.0 mg/l were prepared separately. These supersaturated solutions were stirred for two days to reach maximum solubility. After the stirring period the mixtures contained a test substance floating layer and test substance particles. Collection of the water phase by siphoning or centrifugation was not an option considering the specific gravity of the test substance. After the stirring period the mixtures were therefore filtered through a paper filter (ca.> 5 µm). In addition a ten-fold dilution was prepared from the filtrate prepared at a loading rate of 1.0mg/l. All test solutions were clear and colourless, except for the two highest test concentrations, which were very slight to slightly hazy, respectively.

 

A final test was performed exposing exponentially growing algae to 5µm filtered solutions prepared at loading rates of 1.0, 10 and 100mg/l and a ten-fold dilution of the filtrate prepared at a loading rate of 1.0 mg/l for a period of 72 hours. Samples for analytical confirmation of actual exposure concentrations were taken from all test solutions at the start and at the end of the test.

 

Analysis of the samples showed that the average exposure concentration in the WAF prepared at a loading rate of 100mg/l was at above or approximated the solubility limit of HATCOL 5236 (i.e.<0.2 mg/l). Since toxicological relevant test concentrations could not be analysed, biological results were based on loading rates.

 

The study met the acceptability criteria prescribed by the protocol and was considered valid.

 

Due to the fact that test concentrations below or approximating the water solubility level of HATCOL 5236 could not be analysed as these were below the limit of quantification, a different approach was followed for the calculation of EC and NOEC-values.

To this respect the following text quoted from the OECD guidance on classification of chemicals is applicable: 'Many substances covered by the classification scheme are in fact mixtures, for which measurement of exposure concentrations is difficult, and in some cases impossible. Substances such as petroleum distillate fractions, polymers, substances with significant levels of impurities, etc can pose special problems since the toxic concentration is difficult to define and impossible to verify. Typical testing procedures often rely on the formation of a Water Soluble Fraction (WSF) or Water Accommodated Fraction (WAF) and data are reported in terms of loading rates. These data may be used in applying the classification criteria.' (Organization for Economic Co-operation and Development (OECD), OECD guidance document on the use of the harmonised system for the classification of chemicals which are hazardous for the aquatic environment, OECD series on testing and assessment number 27, July 23, 2001).

 

HATCOL 5236 reduced growth rate of Sefenastrum capricamutum significantly at loading rates of 10 mg/l and higher.

The EL50 for total cell growth inhibition (EBL50:0·72h) was 8.0 mg/l with a 95% confidenceinterval ranging from 3.5 to 19 mg/l.

The EL10 for total cell growth inhibition (EBL10:0·72h) was 0.71 mg/l with a 95% confidence interval ranging from 0.28 to 1.80 mg/l.

 

As growth rate is derived from the slope under the growth curve in a logarithmic plot, themeasure of the specific growth rate is preferable over biomass following from the mathematical nature of exponential growth. In addition total cell growth is depended on growth rate, whereasgrowth rate is independent from cell density during exponential cell growth as was the case inthe present test for all concentrations. For evaluation of the results and for classification of thetest substance it is therefore advised to use growth rate rather than total cell growth.

 

The EL50 for growth rate reduction (ERL50:0-72h) was 47 mg/l with a 95% confidence interval ranging from 9.9 to 230 mg/l.

The EL10 for growth rate reduction (ERL10:0-72h) was 2.0 mg/l with a 95% confidence interval ranging from 0.41 to 9.6 mg/l.

 

The NOELR for both cell growth inhibition and growth rate reduction was 1.0 mg/l.

Description of key information

The read across for substance, CAS: 156558-98-4; EC: 451-190-0; is based upon the analogous substances to which basic form, degree of substitution of functional groups is not considered to effect the proposed read across for the endpoint of toxicity to aquatic algae and cyanobacteria. The NOELR for the substance based on the mean of the information available is deemed to be 25.11 mg/L, to which no adverse effects were noted upto the limit of solubility.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

25.11 mg/L

Additional information

HATCOL 3331

Selenastrum capricornutum, Fresh Water Algal Growth Inhibition Test with HATCOL 3331.

The batch of HATCOL 3331 tested was a clear colourless liquid with a purity of 97.3% and the substance was not completely soluble in test medium at the concentration tested. The water solubility of Hatcol 3331 at 20.3 ± 0.8°C was determined to be < 2.0x10E-4 g/I, according to the flask method (NOTOX Project 365052). The partition coefficient (n-octanol/water), Pow, was determined to be ≥ 5.1*10E6 (log Pow ≥ 6.7) at 20.3 ± 0.8°C (NOTOX Project 365085).

Preparation of test solutions started with a stock solution of 100 mg/l applying 2 days of magnetic stirring to ensure maximum solubility in test medium. The resulting solution was slightly hazy with a floating layer and test substance droplets. Collection of the water phase by siphoning or centrifugation was not an option considering the specific gravity of the test substance. After the stirring period the mixture was therefore filtered through a paper filter(ca. > 5 µm). The filtrate was very slightly hazy.

 

A limit test was performed exposing exponentially growing algal cultures to a filtered (ca. 5µm) HATCOL 3331 solution prepared at a loading rate of 100 mg/I and a blank-control. The initial cell density was 10⁴cells/ml. The total test period was 72 hours. Samples for determination of actual exposure concentrations were taken at the start, after 24 hours of exposure and at the end of the test period.

 

Analysis of the samples showed that average exposure concentrations were at or above the solubility limit of HATCOL 3331 (I.e. < 0.2 mg/l).

 

The study met the acceptability criteria prescribed by the protocol and was considered valid.

 

HATCOL 3331 induced no inhibition of cell growth or reduction of growth rate in Selenastrum capricornutum at the concentration obtained in a filtered solution prepared at a loading rate of 100 mg/l, corresponding to an average exposure concentration of 0.22-0.79 mg/I (NOEC).

Due to the very low solubility of HATCOL 3331 in water, concentration levels that might be toxic for algae could not be reached.

 

HATCOL 3344

Selenastrum capricorutum. Fresh Water Algal Growth Inhibition Test with HATCOL 3344.

HATCOL 3344 is a clear colourless liquid with a purity of 96.9%. The water solubility of HATCOL 3344 at 20.0 ± 0.8°C was determined to be < 2.0x10e-4 g/l (pH was 7.4) using the flask method (NOTOX Project 365535).

 

All test solutions with a loading rate at or above 1.0 mg/l were prepared separately (loading rates of 1.0, 10 and 100 mg/I). These supersaturated solutions were stirred for two days to reach maximum solubility. Collection of the water phase by siphoning or centrifugation was not an option considering the specific gravity of the test substance (see also NOTOX Project 365535). After the stirring period all three mixtures were therefore filtered through a paper filter (Schleicher and Schuell 604, ca. > 5µm). Finally a ten-fold dilution was prepared from the filtrate prepared at a loading rate of 1.0 mg/l. All test solutions were clear and colourless.

 

A final test was performed exposing exponentially growing algae to 5 µm filtered solutions prepared at loading rates of 1.0, 10 and 100 mg/l and in addition to a ten-fold dilution of the filtrate prepared at a loading rate of 1.0 mg/l for a period of 72 hours. Samples for analytical confirmation of actual exposure concentrations were taken from all test solutions at the start, after 24 hours of exposure and at the end of the test.

 

Analysis of the samples showed that the average exposure concentration in the WAFs prepared at loading rates of 10 and 100 mg/l approximated the solubility limit of HATCOL 3344 (i.e. < 0.2 mg/l). However, since not all components were quantifiable in the calibration chromatograms, it was not possible to determine the concentration of the total test substance. Therefore, the toxicological evaluation was based on loading rates.

 

In the controls, cell density increased by an average factor of > 16 within 3 days. Temperature and pH remained within the ranges prescribed by the protocol, except for the pH of the blank-control and the lowest test concentration, which increased above 9.0 at the end of the test period. Since this deviation could be related to a relatively high rate of algal growth it was not considered to have affected the validity criteria of the test.

 

Due to the fact that test concentrations below or approximating the water solubility level of HATCOL 5236 could not be analysed as these were below the limit of quantification, a different approach was followed for the calculation of EC and NOEC-values.

 

HATCOL 3344 reduced growth rate of Selenastrum capricornutum significantly at loading rates of 1.0 mg/l and higher.

The Table below gives the effect parameters based on loading rates.

Parameter	Loading rate HATCOL 3344 (mg/l)	95%-confidence Interval
Observed NOELR	0.1
72h-EBL10	*	*
72h-EBL0	25	19-35
72h ERL10	12	10-15
72h ERL50	>100

*Could not be calculated using log-linear regression.

As growth rate is derived from the slope under the growth curve in a logarithmic plot, the measure of the specific growth rate is preferable over biomass following from the mathematical nature of exponential growth.

 

HATOL 5236

Selenastrum capricomutum, Fresh Water Algal Growth Inhibition Test with HATCOL 5236.  

HATCOL 5236 is a clear pale yellow liquid with a purity of 97.6%. The water solubility of HATCOL 5236 at 20.0± 0.8°C was determined to be< 2.0x10^-4g/l using the flask method (NOTOX Project 365041).

 

All test solutions with a loading rate at or above 1.0 mg/l were prepared separately. These supersaturated solutions were stirred for two days to reach maximum solubility. After the stirring period the mixtures contained a test substance floating layer and test substance particles. Collection of the water phase by siphoning or centrifugation was not an option considering the specific gravity of the test substance. After the stirring period the mixtures were therefore filtered through a paper filter (ca.> 5 µm). In addition a ten-fold dilution was prepared from the filtrate prepared at a loading rate of 1.0mg/l. All test solutions were clear and colourless, except for the two highest test concentrations, which were very slight to slightly hazy, respectively.

 

A final test was performed exposing exponentially growing algae to 5µm filtered solutions prepared at loading rates of 1.0, 10 and 100mg/l and a ten-fold dilution of the filtrate prepared at a loading rate of 1.0 mg/l for a period of 72 hours. Samples for analytical confirmation of actual exposure concentrations were taken from all test solutions at the start and at the end of the test.

 

Analysis of the samples showed that the average exposure concentration in the WAF prepared at a loading rate of 100mg/l was at above or approximated the solubility limit of HATCOL 5236 (i.e.<0.2 mg/l). Since toxicological relevant test concentrations could not be analysed, biological results were based on loading rates.

 

The study met the acceptability criteria prescribed by the protocol and was considered valid.

 

Due to the fact that test concentrations below or approximating the water solubility level of HATCOL 5236 could not be analysed as these were below the limit of quantification, a different approach was followed for the calculation of EC and NOEC-values.

 

HATCOL 5236 reduced growth rate of Selenastrum capricamutum significantly at loading rates of 10 mg/l and higher.

The EL50 for total cell growth inhibition (EBL50:0·72h) was 8.0 mg/l with a 95% confidence interval ranging from 3.5 to 19 mg/l.

The EL10 for total cell growth inhibition (EBL10:0·72h) was 0.71 mg/l with a 95% confidence interval ranging from 0.28 to 1.80 mg/l.

As growth rate is derived from the slope under the growth curve in a logarithmic plot, the measure of the specific growth rate is preferable over biomass following from the mathematical nature of exponential growth. In addition total cell growth is depended on growth rate, whereas growth rate is independent from cell density during exponential cell growth as was the case in the present test for all concentrations. For evaluation of the results and for classification of the test substance it is therefore advised to use growth rate rather than total cell growth.

The EL50 for growth rate reduction (ERL50:0-72h) was 47 mg/l with a 95% confidence interval ranging from 9.9 to 230 mg/l.

The EL10 for growth rate reduction (ERL10:0-72h) was 2.0 mg/l with a 95% confidence interval ranging from 0.41 to 9.6 mg/l.

The NOELR for both cell growth inhibition and growth rate reduction was 1.0 mg/l.

 

One GLP Guideline study conducted according to OECD 201 is available investigating the toxicity of the source substance PE/TMP tetra/tri C5, i-C5, C7, C8, i-C9, C10 ester to freshwater algae (Mead and Mullee, 1998). The green algae Pseudokirchneriella subcapitata was exposed for 96 h to a limit Water Accommodated Fraction (WAF) of nominal 100 mg/L. GC analysis resulted that a measured concentration were below 0.73 mg/L over the time of exposure. No effects on growth and yield of algae were observed resulting in an EL50 (72 h) of > 100 mg/L (nominal). The NOEC (72 h) was derived to be≥100 mg/L (nominal).

Based on the results from a structurally similar source substance (in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5) it can be concluded that Pentaerythritol tetraesters of n-C5, n-C7, n-C8, i-C9 and n-C10 fatty acids will not show effects up to the limit of water solubility.