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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Apr - 24 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 202 (Daphnia sp. Acute Immobilisation Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

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 WAFs (replicates R1 - R2 and R3 - R4 pooled) at 0 and 48 h for quantitative analysis. 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: at ambient temperature in tight and dark conditions for approximately 48 h.

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 immobilisation or adverse reactions to exposure were observed.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Source: in house culture, originally obtained from the Institut National de Recherche Chimique Appliquee (IRCHA), France.
- Age at study initiation (mean and range, SD): < 24 h

ACCLIMATION
- Type and amount of food: mixed algae (predominantly Chlorella spp)
- Feeding frequency: daily

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

217 mg/L (mean)

Test temperature:

21 ± 1 °C

pH:

7.6 (mean)

Nominal and measured concentrations:

100 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Type: closed
- Material, size: 250 mL test vessel
- Aeration: no
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 2


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated tap water
- Total organic carbon: <1.0 mgC/L
- Particulate matter: 0.2 mg/L
- Pesticides: <0.03 μg/L
- Chlorine: 0.11 mg/L
- Alkalinity: 110 mg/L
- Conductivity: 468 μS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: Water temperature was recorded daily throughout the study. Dissolved oxygen concentrations and pH were recorded at the start and termination of the study.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light and 8 h darkness cycle with 20 mins dawn and dusk transition periods


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations of immobile Daphnia were made after 24 and 48 h of exposure.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: control, 10 and 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: no immobilisation

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mortality

Details on results:

no immobilisation

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

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

Remarks:

3 substances available to 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

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Remarks on result:

other: PE/TMP tetra/tri C5, i-C5, C7, C8, i-C9, C10 ester

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mortality

Remarks on result:

other: PE/TMP tetra/tri C5, i-C5, C7, C8, i-C9, C10 ester

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Hatcol 5236

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 1.1 - < 1.4 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Hatcol 3331

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 short-term toxicity to invertebrates. The EC50  for the substance based on the mean of the information available is deemed to be 33.77 mg/L, to which no adverse effects were noted upto the limit of solubility.

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 April 2003 to 27 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 202 (Daphnia sp. Acute Immobilisation Test)

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

During the final test duplicate samples for analysis were taken from all test solutions and the blank-control.
Sampling:
Frequency: at t=Oh and t=48h.
Volume: 5 ml from the approximate centre of the test vessel.
Storage: Not applicable, the samples were analysed on the day of sampling.

Vehicle:

no

Details on test solutions:

The standard test procedures required generation of test solutions which should contain completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that disturb the test system should be prevented (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 Hatcol 3331 at 20.3 ± 0.8°C was determined to be < 2.0x1 0E-4 g/l, 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).
All solutions at loading rates of 1.0 mg/l and higher were prepared separately. Following a short period of ultrasonification (in the final test only), these supersaturated solutions were magnetically stirred for two days to ensure maximum solubility in test medium. The resulting solution was colourless, but contained a 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 mixture was therefore filtered through a paper filter (ca. >.5 µm). The filtrate was clear and colourless. Note that test solutions in the limit test originated from the simultaneously performed acute toxicity study in carp (NOTOX Project 364995).

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th. brood, showing no signs of stress such as mortality > 20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Characteristics: For the test selection of young daphnia with an age of < 24 hours, from parental daphnids of more than two weeks old.

BREEDING
Start of each batch: With new-born animals, i.e. less than 3 days old, by placing about 250 of them into 10 litres of medium in an all-glass culture vessel.
Maximum age of the cultures: 4 weeks
Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
Temperature of medium: 18-22°C
Feeding: Daily, a suspension of fresh water algae.
Medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.- P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

No post exposure observation period specified.

Hardness:

The hardness: 250 mg/l expressed as CaC03 and the pH: 8.0 ± 0.2 after aeration.

Test temperature:

The temperature of the test medium was 21.6°C at the start of the test. The temperature continuously measured in a temperature control vessel varied between 20.2 and 21.2°C during the test, and complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).

pH:

7.6 - 7.8

Dissolved oxygen:

8.3 - 8.9

Salinity:

Not applicable - freshwater test

Nominal and measured concentrations:

Measured concentrations

Details on test conditions:

TEST CONCENTRATIONS
Range: Based on the results of the limit test, filtrates (ca. 5 µm) prepared at loading rates of 1.0, 10 and 100 mg/l, and a 10-fold dilution of the filtrate prepared at 1.0 mg/l.
Controls: Test medium without test substance or other additives (0 mg/I).

TEST PROCEDURE AND CONDITIONS
Test type: Static
Test duration: 48 hours
Test vessels: 100 ml, all-glass
Medium: ISO. prepared in mill-RO water
Number of daphnia: 20 per concentration
Loading: 5 per vessel containing 80 ml medium
Light: 16 hours photoperiod daily
Feeding: No feeding
Aeration: No aeration of the test solutions.
Introduction of daphnia: Within % hours after preparation of the test solutions.

MEASUREMENTS AND RECORDINGS
Immobility: (Including mortality) At 24 hours and 48 hours.
pH and dissolved oxygen: At the beginning and at the end of the test, for all concentrations and the control(s).
Temperature of medium: Continuously in a temperature-control vessel, beginning at the start of the test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate.

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 1.1 - < 1.4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

LIMIT TEST
Measured 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 HATCOL 3331.
The analytical results showed that the filtered solution prepared at a loading rate of 100 mg/l contained an initial concentration of 0.50 mg/l (based on peaks 1 and 2) or 0.48 mg/l (based on peak 3). At the end of the test period the test concentration had decreased below the limit of detection (based on all peaks).
The average exposure concentration, based on peak 1, 2 or 3, respectively, was 0.22, 0.22 or 0.22 mg/l. Hence, the average concentration was above the solubility limit of HATCOL 3331 (i.e. < 0.2 mg/l). The observed decrease was probably a consequence of this extremely low solubility.

Immobility: After 48 hours of exposure, 9 out of 20 daphnids exposed to the filtrate had become immobilised. From 24 hours onwards, a floating layer was observed in the filtrate. The EC50 was expected to approximate the average exposure concentration of 0.22 mg/l.
Based on the results of the limit test, the ECso was expected to approximate the water solubility of HATCOL 3331. The actual concentrations were below 1.0 mg/l, and in fact only slightly higher than the limit of quantification. Therefore, the performance of a full study with filtrates prepared at loading rates increasing step-wise by a factor less than 10 would not add to an accurate determination of an EC50. Following from this, a final study was designed using loading rates spaced by a factor of 10.

FINAL TEST
Measured concentrations: Initial concentrations in the filtrates prepared at 1.0, 10 and 100 mg/l were respectively < 0.1, 2.0 and 1.7 mg/l (based on peak 1), < 0.1, 1.9 and 1.6 (based on peak 2) or < 0.1, 1.9 and 1.6 (based on peak 3). After 48 hours, the concentration in the 1.0 mg/l filtrate had remained below the limit of quantification (for all peaks), while the concentrations in the 10 and 100 mg/l filtrate had decreased respectively to 0.83 and 0.72 mg/l (based on peak 1), 0.96 and 0.88 mg/l (based on peak 2) or 1.1 and 0.99 mg/l (based on peak 3).
The average exposure concentration in the 1.0 mg/l filtrate approximated the water solubility (i.e. < 0.1 mg/l), while average exposure concentrations in the 10 and 100 mg/l filtrates were maintained above the water solubility of HATCOL 3331. This was confirmed by the presence of a floating layer in these solutions (10 and 100 mg/l filtrates).

Immobility: The responses recorded in this test were unexpected considering the results of the limit test. Although the concentration in this test were higher than in the limit test, no immobilisation was observed. Similarly to the limit test, a floating layer was observed in the filtrate prepared at 100 mg/l, as well as in the filtrate prepared at 10 mg/l. At the end of the test, the daphnia exposed to these filtrates were examined under a dissection microscope, and found to be covered by test substance. Such observation was not included in the limit test. Even though test substance was attached to the daphnia, this did not seem to hamper them in their movements and mobility. The reason for the immobility in the limit test remains unclear. However, the results of the final test, in which 2 concentrations tested were above the water solubility limit and 1 concentration approximated this limit, while no significant immobility was observed, are considered sufficient to conclude that the EC50 for immobilisation is above the water solubility of HATCOL 3331. Note that a response of 10% is accepted for the controls and therefore not considered to be related to treatment.

Experimental conditions: These test conditions remained within the limits prescribed by the protocol (pH: 6.0-8.5, not varying by more than 1 unit; oxygen: > 7 mg/l at the start, ≥ 5 mg/l at the end of the test).
The temperature of the test medium was 21.6°C at the start of the test. The temperature continuously measured in a temperature control vessel varied between 20.2 and 21.2°C during the test, and complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).

ACCEPTABILITY OF THE TEST
1. In the control, no daphnia became immobilised or trapped at the surface of the water.
2. The oxygen concentration was > 5 mg/l at the end of the test. Other test conditions (pH, temperature) remained within the limits described by the protocol.
3. The 24h-EC50 (based on initial concentrations) of potassium dichromate was within the range of 0.6 to 1.7 mg/l.

Results with reference substance (positive control):

48-hour Acute Toxicity Study in Daphnia magna with K2Cr2O7 (NOTOX Project 381882).
The study procedures described in this report were based on the ISO International Standard 6341, the EEC directive 92/69, Part C.2. "Acute toxicity for Daphnia", December 1992, and the OECD guideline No. 202: "Daphnia sp., Acute Immobilisation Test", Adopted April 4, 1984.
The reference test was carried out to check the sensitivity of the test system as used by NOTOX. Daphnia were exposed for a maximum of 48 hours to K2Cr2O7 concentrations of 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/l and to a blank control. Ten daphnia were exposed per concentration.
The reference substance, potassium dichromate (K2Cr2O7 art. 4864, batch no. K28974764) was obtained from Merck, Darmstadt, Germany.
The actual responses in this reference test with KzCrz07 are within the ranges of the expected responses at the different concentrations. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at NOTOX.
The 24h-EC50 was 1.2 mg/l with a 95% confidence interval between 1.0 and 1.5 mg/l.
The 48h-EC50 was 0.60 mg/l with a 95% confidence interval between 0.52 and 0.76 mg/l.
The raw data from this study are kept in the NOTOX archives. The test described above was performed under GLP.

Reported statistics and error estimates:

Not specified in the study report.

Table 1: Incidence of immobility in the limit test:

Loading rate HATCOL 3331 (mg/l)	Vessel number	Number Daphnia exposed	Response at 24h^1		Response at 48h
			Number	%	Number	%
Blank-control	A	5	0	0	0	0
	B	5	0		0
	C	5	0		0
	D	5	0		0
100	A	5	1 (2)	5	3	45
	B	5	0		2
	C	5	0 (2)		1
	D	5	0		3

¹Between brackets: number of daphnids trapped at the surface. These organisms were reimmersed into the solution before recording of mobility.

 

Table 2: Average exposure concentrations during the final test

Loading rate HATCOL 3331 (mg/l)	Concentration, mg/l
	Based on peak 1			Based on peak 2			Based on peak 3
	t=0	t=48	Average	t=0	t=48	Average	t=0	t=48	Average
0.10	0.050^1	n.d.	n.a.	0.050^1	n.d.	n.a.	0.050^1	n.d.	n.a.
1.0	0.050^1	0.050^1	0.050	0.050^1	0.050^1	0.050	0.050^1	0.05^1	0.050
10	1.97	0.83	1.3	1.94	0.96	1.4	1.90	1.05	1.4
100	1.66	0.72	1.1	1.62	0.88	1.2	1.60	0.99	1.3

n.d., not detected; n.a., not applicable

¹measured concentration was below limit of quantification; therefore set at LOQ/2, i.e. 0.050 mg/l

 

Table 3: Acute immobilisation of daphnia after 24 and 48 hours in the final EC50 test

Loading rate HATCOL 3331 (mg/l)	Average concentration (mg/l)	Vessel number	Number Daphnia exposed	Response at 24h^1		Response at 48 h^1
				Number	%	Number	%
Blank-control	-	A	5	0 (1)	0	0	0
		B	5	0		0
		C	5	0		0
		D	5	0		0
0.10	-	A	5	0	0	0	0
		B	5	0		0
		C	5	0		0
		D	5	0 (1)		0
1.0	< 0.1	A	5	0	0	0	0
		B	5	0 (2)		0
		C	5	0		0
		D	5	0		0
10^2	1.3-1.4	A	5	0 (5)	0	0^3	0
		B	5	0 (5)		0^3
		C	5	0 (5)		0 (1)^3
		D	5	0 (5)		0^3
100^2	1.1-1.3	A	5	0 (5)	0	0^3	5
		B	5	0 (5)		0 (1)^3
		C	5	0 (5)		1^3
		D	5	0 (5)		0^3

¹between brackets: number of daphnia trapped at the surface. These organisms were reimmersed into the respective solutions before recording of mobility.

²a floating layer was observed starting at 24 h

³all daphnids were observed to be covered with test substance.

 

Table 4: pH and oxygen concentrations during the final test

Loading rate HATCOL 3331 (mg/l)	Average concentrations (mg/l)	Start (t=0 h)		End (t=48 h)
		pH	O2	pH	O2
Blank-control	-	7.7	8.3	7.7	8.9
0.10	-	7.7	8.3	7.8	8.8
1.0	< 0.1	7.7	8.3	7.8	8.8
10	1.3-1.4	7.6	8.3	7.8	8.8
100	1.1-1.3	7.6	8.4	7.7	8.7

 

Acute immobilization of daphnia after 24 and 48 hours in the reference test with potassium dichromate:

Concentration (mg/l)	Number Exposed	% immobile		Expected response (%) After 48 hours^1
		24 h	48 h	Minimal	Maximal
Blank-control	10	0	0	0	10^2
0.10	10	0	0	0	10
0.18	10	0	0	0	10
0.32	10	0	0^3	0	30
0.56	10	0	30	0	100
1.0	10	10	100	40	100
1.8	10	100	100	100	100

¹Based on historical data of the previous years (n>60)

²A maximum response of 10% does not invalidate the results of the test

³One daphnia was observed trapped at the surface of the test solutions. This daphnid was reimmersed in the respective solutions before scoring of mobility.

Validity criteria fulfilled:

yes

Conclusions:

Due to the very low solubility of HATCOL 3331 in water, concentration levels that might be toxic for daphnia could not be reached. Therefore, the 48h-EC50 exceeded the maximum solubility of HATCOL 3331 in test medium.

Executive summary:

Acute Toxicity Study in Daphnia magna with HATCOL 3331.

The study procedures described in this report were based on the ISO International Standard6341: 'Water quality - Determination of the inhibition of the mobility of Daphnia magna Straus-Acute toxicity test, Third edition, 1996-04-01. In addition, the procedures were designed to meet the test methods and validity criteria of the EEC directive 92/69, Part C: Methods for the determination of ecotoxicity, Publication No. L383, December 1992, C.2. "Acute Toxicity for Daphnia", and the OECD guideline No. 202 Part I: "Daphnia sp., Acute Immobilisation Test", Adopted April 4, 1984.

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 watersolubilityof Hatcol 3331 at 20.3 ± 0.8°C was determined to be < 2.0x10E-4 g/l, according to the flask method (NOTOX Project 365052). The partition coefficient (n-octanol/water), Pow, wasdetermined to be ≥ 5.1*10E6 (log Pow ≥ 6.7) at 20.3 ± 0.8°C (NOTOX Project 365085).

All solutions at loading rates of 1.0 mg/l and higher were prepared separately. These supersaturated solutions were magnetically stirred for two days to ensure maximum solubility in test medium. The resulting solution was colourless, but contained a 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 mixture was therefore filtered through a paper filter (ca. > 5 µm). The filtrate was clear and colourless.

The project was started with a limit test, exposing daphnia to a filrate (ca. > 5 µm) prepared at a loading rate of 100 mg/l and a blank-control. The test was performed in duplicate with 5 daphnids per vessel and samples for analysis were taken at the start and the end of the test. The analytical results showed that the average exposure concentration was higher than the solubility limit of HATCOL 3331 (i.e. < 0.2 mg/l). At the end of the test, 45% of the daphnids exposed to the filtrate were immobilized. Since concentrations were all below 1.0 mg/l, a final test was performed using a range of concentrations nominally spaced by a factor of 10.

A final test was performed exposing daphnia to filtrates (ca. > 5 µm) prepared at loading rates of 1.0, 10 and 100 mg/l and a 10-fold dilution of the filtrate at 1.0 mg/l. Samples for analysis taken at the start and the end of the test showed that the average exposure concentration in the 1.0 mg/l filtrate approximated the water solubility (i.e. < 0.2 mg/l), while average exposure concentrations in the 10 and 100 mg/l filtrates were maintained above the water solubility of HATCOL 3331.

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

HATCOL 3331 did not induce acute immobilisation of Daphnia magna at the concentration obtained in a filtered solution prepared at a loading rate of 100 mg/l, corresponding to an average exposure concentration above the water solubility, i.e. 1.1 -1.4 mg/l after 48 hours of exposure (NOEC). Also, no immobilisation was observed at concentrations approximating the water solubility.

In conclusion: Due to the very low solubility of HATCOL 3331 in water, concentration levels that might be toxic for daphnia could not be reached. Therefore, the 48h-EC50 exceeded the maximum solubility of HATCOL 3331 in test medium.

Reference 4

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 March 2003 to 26 June 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 202 (Daphnia sp. Acute Immobilisation Test)

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

During the limit test duplicate samples were taken from the blank-control and the filtered solution prepared at a loading rate of 100 mg/l for analysis. The method of analysis is described in the appended Analytical Report.
Sampling:
Frequency: At t=0 and t=48h
Volume: 10 ml from the approximate centre of the test vessels.
Storage: Not applicable, samples were analysed on the day of sampling.
Additionally, the filter used preparation of the test concentration was stored in a freezer for possible analysis in case no test concentrations could be analysed in the samples taken at the start of the test.

Vehicle:

no

Details on test solutions:

The standard test procedures required generation of test solutions that contain completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions.
The testing of concentrations that disturbed the test system were prevented (e.g. film of the test substance on the water surface).
HATCOL 5236 is a clear colourless liquid with a purity of 96.7%. 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).
A stock solution was prepared at a loading rate of 100 mg/l. This supersaturated solution was stirred for two days to reach maximum solubility. After the stirring period the mixture was hazy and contained a test substance floating layer. 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 filer (Schleicher and Schuell 604) to remove the larger undissolved test substance particles (ca. > 5µm). The filtrate was still slightly hazy.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%. presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Characteristics: For the test selection of young daphnia with an age of <24 hours, from parental daphnids of more than two weeks old.

BREEDING
Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 10 litres of medium in an all glass culture vessel.
Maximum age of the cultures: 4 weeks
Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
Temperature of medium: 18-22°C
Feeding: Daily, a suspension of fresh water algae.
Medium: M7, as prescribed by Dr. Elendt-Schneider.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

No post exposure observation period specified.

Hardness:

The hardness: 250 mg/l expressed as CaCO3 and the pH: 8.0 ± 0.2 after aeration.

Test temperature:

The temperature of the test medium was 20.8°C at the start of the test. The temperature continuously measured in a temperature control vessel varied between 20.0 and 21.3°C during the test.

pH:

pH: 6.0-8.5, not varying by more than 1 unit

Dissolved oxygen:

oxygen: >7 mg/l at the start, ≥5 mg/l at the end of the test

Salinity:

Not applicable - freshwater study

Nominal and measured concentrations:

Measured concentrations
The test substance is a substituted polymer, resulting in a number of peaks in the chromatogram of the test substance solutions. Quantification was based on the two largest peaks. The analytical results showed that the filtered solution prepared at a loading rate of 100 mg/l had an initial concentration of 5.6 mg/l (when based on peak 1) or 5.2 mg/l (when based on peak 2). After 48 hours of exposure the test concentration had decreased to 0.97 mg/l (when based on peak 1) or 0.75 mg/l (when based on peak 2). Hence, concentrations remained above the solubility limit of HATCOL 5236 (i.e. < 0.2 mg/l throughout the test. The observed decrease was probably a consequence of this extremely low solubility.
The average exposure concentration. based on peak 1, was 2.3 mg/l, while the average exposure concentration, based on peak 2, was 2.0 mg/l.

Details on test conditions:

TEST CONCENTRATIONS
HATCOL 5236: A 5 µm filtrate of a supersaturated solution prepared at a loading rate of 100 mg/l.
Controls: Test medium without test substance or other additives (Blank-control).

TEST PROCEDURE AND CONDITIONS
Test type: Static
Test duration: 48 hours
Test vessels: 100 ml, all-glass
Medium: ISO
Number of daphnia: 20 per concentration
Loading: 5 per vessel containing 80 ml medium
Feeding: No feeding
Light: 16 hours photoperiod daily
Aeration: No aeration of the test solutions.
Introduction of daphnids Within 20 minutes after preparation of the test solutions.

MEASUREMENTS AND RECORDINGS
Immobility (including mortality): At 24 hours and 48 hours.
pH and dissolved oxygen: At the beginning and at the end of the test, for the limit concentration and the control.
Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Immobility
At the end of the test period undissolved test substance particles were observed in the test concentration, which was due to testing above water solubility. Daphnids however were not disturbed by this precipitation.

Experimental conditions
These test conditions remained within the limits prescribed by the protocol (pH: 6.0-8.5, not varying by more than 1 unit; oxygen: > 7 mg/l at the start, ≥5 mg/l at the end of the test).
The temperature of the test medium was 20.8°C at the start of the test. The temperature continuously measured in a temperature control vessel varied between 20.0 and 21.3°C during the test, and complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).

Results with reference substance (positive control):

The actual responses in the reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at NOTOX.
The 24h-EC50 was 1.0 mg/l with a 95% confidence interval between 0.94 and 1.2 mg/l.
The 48h-EC50 was 0.74 mg/l with a 95% confidence interval between 0.66 and 0.84 mg/l.

Reported statistics and error estimates:

ACCEPTABILITY OF THE TEST
1. In the control, no daphnia became immobilised or trapped at the surface of the water.
2. The oxygen concentration was ≥ 5 mg/l at the end of the test. Other test conditions (pH and temperature) were maintained within the limits prescribed by the guidelines.
3. The 24h-EC50 (based on the initial concentration) of potassium dichromate was within the range 0.6 mg/l to 1.7 mg/l.

Incidence OF Immobility in the limit test

Loading rate HATCOL 5236 (mg/l)	Vessel number	Number Daphnia exposed	Response at 24h		Response at 48h
			Number	Total %	Number	Total %
Blank-control	A	5	0	0	0	0
	B	5	0		0
	C	5	0		0
	D	5	0		0
100	A	5	0	0	0*	0
	B	5	0		0*
	C	5	0		0*
	D	5	0		0*

*Precipitation was observed.

 

pH and oxygen concentrations during the limit test

Loading rate HATCOL 5236 (mg/l)	Start (t=0h)		End (t=48h)
	pH	O2	pH	O2
Blank-control	7.9	8.5	8.0	8.7
100	7.8	8.0	7.7	8.1

 

Acute immobilization of daphnia after 24 and 48 hours in the reference test with potassium dichromate:

Concentration (mg/l)	Number exposed	% immobile		Expected response (%)
		24 h	48 h	After 48 hours^1
				Minimal	Maximal
Blank-control	20	0	0	0	10^2
0.10	20	0	0	0	10
0.18	20	0	0	0	10
0.32	20	0	0	0	30
0.56	20	0	5	0	100
1.0	20	35	95	40	100
1.8	20	100	100	100	100

¹Based on historical date of the previous years (n>60).

²A maximum response of 10% does not invalidate the results of the test.

Validity criteria fulfilled:

yes

Conclusions:

Owing to the extremely low solubility of HATCOL 5236 in water, concentration levels toxic for crustaceans could not be reached. Therefore, the 48h-EC50 for Daphnia magna exceeded the maximum solubility of HATCOL 5236 in water.

Executive summary:

Acute Toxicity Study in Daphnia magna with HATCOL 5236.

The study procedures described in this report were based on the ISO International Standard6341: "Water quality- Determination of the inhibition of the mobility of Daphnia magna Straus-Acute toxicity test, Third edition, 1996-04-01. In addition, the procedures were designed to meet the test methods and validity criteria of the EEC directive 92/69, Part C: Methods for the determination of ecotoxicity, Publication No. L383, December 1992, C.2. "Acute Toxicity for Daphnia", and the DECO guideline No. 202 Part I: "Daphnia sp., Acute Immobilisation Test., Adopted April 4, 1984.

HATCOL 5236 is a clear colourless 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.

A stock solution was prepared at a loading rate of 100 mg/l. This supersaturated solution wasstirred for two days to reach maximum solubility. Afterthe stirring period the mixture was hazy and contained a test substance floating layer. 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 filer (ca. > 5µm). The filtrate was still slightly hazy.

A limit test was performed exposing twenty daphnids per concentration to a 5 pm filtered solution prepared at a loading rate of 100 mg/l and a blank-control for a maximum of 48 hours.

The test was performed in quadruplicate with 5 daphnids per vessel. Samples for analytical confirmation of actual exposure concentrations were taken at the start and at the end of the test.

Analysis of the samples showed that concentrations remained above the solubility limit of HATCOL 5236 (i.e. < 0.2 mg/l) throughout the test.

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

HATCOL 5236 did not induce acute immobilisation of Daphnia magna when exposed to a filtered solution prepared at a loading rate of 100 mg/l, corresponding with concentrations abovethe water solubility.

In conclusion: Owing to the extremely low solubility of HATCOL 5236 in water, concentration levels toxic for crustaceans could not be reached. Therefore, the 48h EC50 for Daphnia magna exceeded the maximum solubility of HATCOL 5236 in water.

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 short-term toxicity to invertebrates. The EC50  for the substance based on the mean of the information available is deemed to be 33.77 mg/L, to which no adverse effects were noted upto the limit of solubility.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

33.77 mg/L

Additional information

HATCOL 3331

Acute Toxicity Study in Daphnia magna 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/l, 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).

All solutions at loading rates of 1.0 mg/l and higher were prepared separately. These supersaturated solutions were magnetically stirred for two days to ensure maximum solubility in test medium. The resulting solution was colourless, but contained a 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 mixture was therefore filtered through a paper filter (ca. > 5 µm). The filtrate was clear and colourless.

The project was started with a limit test, exposing daphnia to a filtrate (ca. > 5 µm) prepared at a loading rate of 100 mg/l and a blank-control. The test was performed in duplicate with 5 daphnids per vessel and samples for analysis were taken at the start and the end of the test. The analytical results showed that the average exposure concentration was higher than the solubility limit of HATCOL 3331 (i.e. < 0.2 mg/l). At the end of the test, 45% of the daphnids exposed to the filtrate were immobilized. Since concentrations were all below 1.0 mg/l, a final test was performed using a range of concentrations nominally spaced by a factor of 10.

A final test was performed exposing daphnia to filtrates (ca. > 5 µm) prepared at loading rates of 1.0, 10 and 100 mg/l and a 10-fold dilution of the filtrate at 1.0 mg/l. Samples for analysis taken at the start and the end of the test showed that the average exposure concentration in the 1.0 mg/l filtrate approximated the water solubility (i.e. < 0.2 mg/l), while average exposure concentrations in the 10 and 100 mg/l filtrates were maintained above the water solubility of HATCOL 3331.

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

HATCOL 3331 did not induce acute immobilisation of Daphnia magna at the concentration obtained in a filtered solution prepared at a loading rate of 100 mg/l, corresponding to an average exposure concentration above the water solubility, i.e. 1.1 -1.4 mg/l after 48 hours of exposure (NOEC). Also, no immobilisation was observed at concentrations approximating the water solubility.

In conclusion: Due to the very low solubility of HATCOL 3331 in water, concentration levels that might be toxic for daphnia could not be reached. Therefore, the 48h-EC50 exceeded the maximum solubility of HATCOL 3331 in test medium.

 

HATCOL 5236

Acute Toxicity Study in Daphnia magna with HATCOL 5236.

HATCOL 5236 is a clear colourless 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.

A stock solution was prepared at a loading rate of 100 mg/l. This supersaturated solution was stirred for two days to reach maximum solubility. After the stirring period the mixture was hazy and contained a test substance floating layer. 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 filer (ca. > 5µm). The filtrate was still slightly hazy.

A limit test was performed exposing twenty daphnids per concentration to a 5 pm filtered solution prepared at a loading rate of 100 mg/l and a blank-control for a maximum of 48 hours.

The test was performed in quadruplicate with 5 daphnids per vessel. Samples for analytical confirmation of actual exposure concentrations were taken at the start and at the end of the test.

Analysis of the samples showed that concentrations remained above the solubility limit of HATCOL 5236 (i.e. < 0.2 mg/l) throughout the test.

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

HATCOL 5236 did not induce acute immobilisation of Daphnia magna when exposed to a filtered solution prepared at a loading rate of 100 mg/l, corresponding with concentrations above the water solubility.

In conclusion: Owing to the extremely low solubility of HATCOL 5236 in water, concentration levels toxic for crustaceans could not be reached. Therefore, the 48h EC50 for Daphnia magna exceeded the maximum solubility of HATCOL 5236 in water.

Since no studies investigating the short-term toxicity of Pentaerythritol tetraesters of n-C5, n-C7, n-C8, i-C9 and n-C10 fatty acids (EC 451-190-0) to aquatic invertebrates are available for this endpoint, in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5 a read across to the structurally related source substance PE/TMP tetra/tri C5, i-C5, C7, C8, i-C9, C10 ester was conducted. The source substance is characterized as an ester of pentaerythritol/trimethylolpropane with fatty acid C-chains of C5, C7, C8, C9iso and C10 whereas the target substance is solely a pentaerythritol ester but esterified with fatty acids of the same C-chain lengths and a slightly different composition. Due to the very similar composition of both substances the source substance can thus be used for read-across to evaluate the toxicity of the target substance.

One GLP Guideline study according to OECD 202 is available investigating the short-term toxicity of the source substance PE/TMP tetra/tri C5, i-C5, C7, C8, i-C9, C10 ester to freshwater fish (Wetton and Mullee, 1998). Daphnia magna was exposed for 48 h to a limit Water Accommodated Fraction (WAF) of nominal 100 mg/L. GC analysis resulted in a measured initial concentration of 0.40 mg/L. No effects on mobility were observed resulting in an EL50 (48 h) of > 0.40 mg/L (measured initial concentration) and > 100 mg/L (nominal), respectively.

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.