N,N',N'',N'''-tetrakis(4,6-bis(butyl-(N-methyl-2,2,6,6-tetramethylpiperidin-4-yl)amino)triazin-2-yl)-4,7-diazadecane-1,10-diamine

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Mar. 3, 1999 to Feb. 28, 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study with restrictions - the analytical recoveries of the test substance concentration of at test start and test end revealed partly strong variations - dose-response relationship inconsistent

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 0.3, 1, 3, 10, 30 and 100 mg/l
- Sampling method: During the final test samples were taken from each concentration before introduction of algae at the start of the exposure for analysis. At the end of the exposure, the replicates incubated without algae were pooled at each concentration and the pooled solution will be used for analysis.
- Sample storage conditions before analysis: stored in a deep freeze.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
All test solutions were prepared separately and mixed with test medium for a defined period. Subsequently, the test solutions were allowed to stabilize. The water fractions were then centrifuged (at 49,000 g for 15 minutes) to remove any undissolved particles. The resulting water phases were defined as water accommodated fractions (WAFs) prepared at a nominal loading.
Available data showed that adsorption to glass was a major cause for loss of test substance concentration. Therefore, the range-finding test was repeated with glassware silanised applying a 2% dichlorodimethylsilane solution in hexane. The glassware was dried in a kiln prior to silanisation. Subsequently, the glassware were rinsed with small amounts of the silanisation solution. Then, the glassware were rinsed with hexane to remove any surplus of dichlorodimethylsilane. The hexane was removed by evaporation
applying air under pressure. Then all glassware was rinsed three times with deionized water and three times with the sterilised algal medium.

Controls
1. Test medium without test substance or other additives (blank control).
2. Test medium without test substance or other additives but treated in the same way as the solutions treated with test substance in silanised glass ware (treatment control).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Selenastrum capricornutum
- Strain: CCAP 278/4.
Cell density A volume of 0.22 ml of an algal suspension was added to each replicate providing a cell density of 10000 cells/ml.
During incubation the algal cells were kept in suspension by continuous shaking.
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 (4000-9000 lux) in a climate room at a temperature of 23 ± 2°C.
Pre-culture 3 or 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 2.10^ cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured directly before use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/l

Test temperature:

22-23ºC

pH:

7.9 - 9.3
Loading (mg/L) pH, 0 hours exposure pH, 72 hours exposure
0 (treatment control) 7.9 9.1
0.3 8 9.2
1 7.9 9.3
3 7.8 9.3
10 8.1 8
30 7.9 8
100 7.9 8.1
0 (blank control) 8.3 8.6

There was a pH shift during the test from 7.9 to 9.3 which is still in the range that is acceptable according to OECD TG 201. The test item has a calculated pka value of 9.4. In other words, this means that at a pH of 9.4 theoretically there would be an equilibrium of ionized and non-ionized species of the test item which would alter the bioavailability during the test. Therefore, the majority of the test item was still present in the hydrogenated form of the molecule and the theoretical bioavailability was not significantly changed. Leading to the conclusion, that the change in the pH value during the test, which is rather normal and speaks for a “helathy” test system, did not influence the results. Taking further into account that the toxicity is mostly due to the residues and impurities this fact becomes even less important.

Salinity:

freshwater

Nominal and measured concentrations:

See 'any information on results incl tables', Table1

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml, all-glass; it is implied in the report that the final test glassware is silanised, but not actually stated.
- Initial cells density: 10 000 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6 (50mL lank and treatment control with algae),
- 5 replicates of 100mL of each test concentration without algae


GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- M2-medium according to the ISO-Standard "Algal growth inhibition test" Nov. 1989; formulated using Milli-Q water preventing precipitation and with the following composition:
NH4CI 15 mg/l
MgCl2.6H2O 12 mg/l
CaCl2.2H20 18 mg/l
MgS04.7H20 15 mg/l
KH2P04 1.6 mg/l
FeCl3.6H20 80 µg/l
Na2EDTA.2H20 100 µg/l
H3BO3 185 µg/l
MnCl2.4H20 415 µg/l
ZnCl2 3 µg/l
CoCls.SHzO 1.5 µg/l
CuCl2.2H20 0.01 µg/l
Na2Mo04.2H20 7 µg/l
NaHCO3 50 mg/l
Hardness (Ca+Mg) 0.24 mmol/l (24 mg CaCOs/l)
- Source/preparation of dilution water: Milli-Q water (Tap water purified by reverse osmosis and then passed over activated carbon and ion-exchange cartridges (Millipore Corp., Bedford, Mass., USA).
- Intervals of water quality measurement: pH at 0 and 72 hours and temperature every day (temperature-control vessel).

OTHER TEST CONDITIONS
- Photoperiod: continuous TLD-lamps of the type 'GroLux' of 30 Watt (Sylvania)
- Light intensity and quality: 3500-3700 lux with a light intensity within the range of 60 to 120 microE.m-2.s-2, not varying by more than 20%.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber at 24-hour intervals
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 Lambda Spectrophotometer (Perkin-Elmer, Illinois, USA), with a cuvette of 5 cm path-length. Algal medium was used as blank.

TEST CONCENTRATIONS
- Water accommodated fractions were prepared at 0.3, 1,3, 10, 30 and 100 mg/l. Spacing factorfactor of 3
- Controls: 1. Test medium without test substance or other additives (blank control). 2. Test medium without test substance or other additives but treated in the same way as the solutions treated with test substance in silanised glass ware
(treatment control).

RANGE FINDING TEST
Two range-finding tests preceded the final test to provide information about the range of concentrations to be used in the final test. In both cases algal suspensions were exposed to water fractions 0.1, 1, 10 and 100 mg/L (nominal)

First range finding test:
normal glassware and water fractions were prepared at 10 and 100 mg/l
After centrifuging, the water fraction prepared at 10 mg/l was used to prepare the water fractions of 0.1 and 1 mg/l. Samples for analysis were taken from the water fraction prepared at 100 mg/l.
Results:
The extinction values measured during the first range-finding test (720 nm) in the WAF prepared at 100 mg/l, indicated almost 50% (control 72h 209-286, 100mg/L 72h 127-165)
reduction of algal growth. The concentration of CAS 106990-43-6 measured in the samples taken from the WAF prepared at 100 mg/l varied from 0.093 mg/l at the start of the test to 0.074 mg/l at the end of the test. These measured concentrations were far below 10 mg/l. However, extinctions were only slightly reduced in the WAF prepared at 10 mg/l in comparison to those of the control. These results indicate that the effect on algal growth was probably not caused by exposure to the main component.

Second range finding test:
The second range-finding test was performed with silanised glass ware and water fractions were prepared at 1 mg/l (5 mg in 5 litres), 10 mg/l (20 mg in 2 litres) and 100 mg/l (200 mg in 2 litres). The 10-mg/l was vigorously mixed to achieve a complete suspension and 50 ml was taken from the suspension and mixed with 5 litres of medium to achieve a water fraction prepared at 0.1 mg/l. After stirring for 72 hours, the solutions were allowed to stabilise overnight. Then volumes of 1500 ml were taken from the water fractions and separated in volumes of ca. 250 ml, which were centrifuged. The supematants were pooled before taking three samples of 500 ml. One sample was used for analysis at the start of the exposure. The other sample was
used to prepare the algal suspensions for exposure. The third sample was used for incubation of replicates under test conditions but without algal cells present. Volumes of 50 ml were added to each replicate of the respective test concentration. The samples taken from each concentration without algae present were analysed at the start of the exposure. At the end of the exposure, the replicates without algae were pooled at each concentration and used for analysis. In addition the replicates at 0.1 mg/l with algae were also pooled and used for analysis.
At the end of the test the replicates of the concentration with an effect near the EC50 were used for additional incubation during two days in clear test medium to examine possible recovery. The volumes were transferred to centrifuge tubes and the algal cells present were separated from the test solution by centrifuging at ca. 3000 g. The supernatant was discarded and the algal cells were resuspended in fresh test medium. The suspensions were transferred to clean vessels and incubated for another 48 hours.
Results:
The concentrations measured in the samples taken from the WAFs prepared at of 1 and 100 mg/l were within the expected range, i.e. 0.007 and 0.850 mg/l, respectively. However, a high recovery of 63% was found in the sample taken at 10 mg/l. This may indicate that the centrifuging of the sample prior to analysis had not been complete. The analysis of the samples taken after 72 hours showed a further decrease of the measured concentration at the nominal level of 100 mg/l. The concentration measured in the WAF prepared at 10 mg/l had decreased to a value within the expected range, i.e. 0.15 mg/l.
Cell densities were slightly lower than the controls in the WAFs of 0.1 and 1.0 mg/l. The high exposure at 10 mg/l as indicated by the analytical results (6.35-0.15 mg/l) was not confirmed in the algal test, since the algal growth was significantly less affected (72h, 71.8 x 10000cells/ml) than recorded at 100 mg/l (72h 32.7 x 10000cells/ml).
The mean areas under the growth curves found for the WAFs prepared at 0.1 and 1.0 mg/l were in the same range and both about 20% lower than the mean area of the treatment control. The values for growth rate show that the effect on algal growth was mainly recorded during the first 48 hours of exposure. The inhibition of total cell growth appears to be treatment related at nominal levels of 10 and 100 mg/l. Growth rate recovered also at these levels.
The algal suspensions incubated at 100 mg/l were centrifuged at the end of the test and the algal pellet was resuspended in fresh algal medium. These algal suspensions were incubated for another 48 hours. The growth rate parameter \i was 0.05357 for the first 24 hours and 0.04962 for the whole 48-hour period. Hence, these values are in the same range as found for the 0-72h interval during exposure to the WAF prepared at 100 mg/l. This indicates no complete recovery of algal growth in fresh medium after exposure to the WAF prepared at 100 mg/l.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.432 mg/L

95% CI:

>= 4.068 - <= 10.728

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: see remarks, results based on re-evaluation from 2017 (106993-43-6_Algae_RC-NOTOXÜROJECT_2017-10-11measured.pdf)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.687

95% CI:

>= 0.102 - <= 1.252

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: see remarks, results based on re-evaluation from 2017 (106993-43-6_Algae_RC-NOTOXÜROJECT_2017-10-11measured.pdf)

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 3.3 - 35 mg/L

Remarks:

WAF

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 31 - 340 mg/L

Remarks:

WAF

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

103.941 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: see remarks

Remarks:

based on re-evaluation 95%-CL 68.2-223.1 WAF

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

7.804 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: see remarks

Remarks:

based on re-evaluation 95%, CL 2.1-14.3 WAF

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence interval: 1.4 - 24 mg/L

Remarks:

WAF

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence interval: 3.9 - 69 mg/L

Remarks:

WAF

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.26 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence interval: 0.03 - 2.3 mg/L

Remarks:

WAF

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.56 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 0.19 - 1.7 mg/L

Remarks:

WAF

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 2.2 - 20 mg/L

Remarks:

WAF

Details on results:

- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: no
- Effect concentrations exceeding solubility of substance in test medium: The concentrations measured in the WAFs prepared at 30 and 100 mg/l exceeded the expected solubility level of ≤ 2 mg/l indicates the presence of an undissolved fraction. This undissolved fraction may have significantly contributed to the effects on algal growth by causing possible mechanical damage.

Measured test substance concentrations
The concentrations measured in the WAFs prepared at 0.3, 3, 10 and 30 mg/l were consistent between t=Oh and t=72h. The last column lists the average concentrations for the total test period of 0-72h. The concentration measured in the WAF prepared at 1 mg/l was below detection at t=0 h, but a detectable level of 0.0562 mg/l was found at t=72 h. Incidentally low recoveries were found in the extracts of the samples. Hence, it is possible that the first measurement was false due to incomplete extraction of CAS 106990-43-6 out of the water phase. Therefore, the exposure concentration was considered to be 0.0562 mg/l for the total test period. The concentration measured in the WAF prepared at 100 mg/l at t=Oh (0.796 mg/l) was lower than the concentration measured in the WAF prepared at 30 mg/l (3.22 mg/l). However, the concentration measured at the end of the test period was in the range of those measured in the WAF prepared at 30 mg/l (4.39 mg/l). Therefore the exposure concentration was considered to be 4.39 mg/l for the total test period. Note that the concentrations measured in the WAFs prepared at 30 and 100 mg/l exceeded the expected solubility level of s 2 mg/l.


The test item has a very low water solubility and from structural properties hydrolysis is not expected. Furthermore, the test item is a large molecule with a molecular weight of 2258.68 g/mol and a diameter of 2.98 nm (Chemsketch version 10.6 (ACD/Labs, 2006) it is well above the threshold values of 1100 g/mol and 1.7 nm outlined in the guidance R.11 for substances that can permeate membranes. Additionally, the test item has a Koc of >662 to > 4682 and adsorbs to solid phase which lowers the bioavailability even further.
Taking all these information into account it is proven that the used test item in the algal study can be seen as worst-case scenario for the test item. The new composition consists only of three substances. Therefore, if the toxicity is due to an impurity which is no longer present the results can be seen as worst-case assumptions. Therefore, the used ECx and NOEC values can be seen as very conservative. Finally, the performance of a new study can be omitted.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
Study period: October1999, Project No 267514
test concentrations: 0.18, 0.32, 0.56, 1.0, 1.8 mg/l and 3.2 mg/l and blank-control
Initial cell density 10000 cells/ml
EbC50 (0-72h): 0.55 mg/L
ErC50 1.5mg/L

Reported statistics and error estimates:

The EC-values with respective 95% confidence intervals have been calculated from the curves for growth inhibition and growth rate reduction versus the log of the nominal loadings.

Table 1: The average measured concentrations of the selected intem

 

WAF at x mg/l	Measured concentration
	t=0	t=72	0-72h
0.3	0.135	0.147	0.141
1	n.d.	0.0562	0.0562*
3	0.0602	0.0555	0.0579
10	0.424	0.408	0.416
30	3.22	3.00	3.11
100	0.796	4.39	4.39*

n.d. = below detection       

*The average concentration was based on the highest value measured, because recovery experiments incidentally showed low recoveries of test substances in the analytical extracts of the samples.

Table 2: Mean cell densities during the final test

WAF prepared at x mg/l	Concentration CAS 106990-43-6 (mg/l)	Mean cell densities (1.0 x 10000 cells/ml) at
		0h	24h	48h	72h
0 (treatment)	-	1.0	8.2	34.5	151.0
0.3	0.141	1.0	8.9	47.0	184.1
1	0.0562	1.0	7.8	31.3	117.8
3	0.0579	1.0	8.2	39.9	163.2
10	0.416	1.0	6.5	25.9	120.4
30	3.11	1.0	4.1	11.1	26.5
100	4.39	1.0	3.6	9.6	14.6
0 (blank)	-	1.0	6.7	38.1	175.3

The extinctions measured in the first replicate of the treatment control hardly increased, which indicated almost no algal growrth. The data of this replicate was excluded from further calculations. The cell densities measured in the other replicates varied from 110 to 210 x l0000 cells/ml, with an average of 151 x l0000 cells/ml. A relative high variation between cell densities was also recorded in the WAF prepared at 1 mg/l.

Table 3: Percentage inhibition during algae test

 

WAF at x mg/l	Measured concentration mg/l	Inhibition (%)
0	-	-12.3
0.3	0.141	-25.7
1	0.0562	17.5
3	0.0579	-9.9
10	0.416	22.1
30	3.11	77.6
100	4.39	84.5

The mean areas calculated for the WAFs prepared at 0.3 and 3 mg/l were in the same range as the mean area of the untreated control (blank) and higher than the mean area of the treatment

controls. An increasing effect on cell growth started at the WAF prepared at 10 mg/l. Based on measured CAS 106990 -43 -6 concentrations, the actual concentrations of 0.0579 (WAF prepared at 3 mg/l) and 0.141 mg/l (WAF prepared at 0.3 mg/l) did not induce inhibition of cell growth. The extent of inhibition recorded at 0.0562 (WAF prepared at 1 mg/l) is probably not related to exposure to the main constituent.

Table 4: Percentage reduction of growth rate at different time intervals during the final test.

WAF prepared at x mg/I	Concentration (mg/l)	Mean growth rate
		µ (0-24 h)	Reduction (%)	µ (0-48 h)	Reduction (%)	µ (0-78 h)	Reduction (%)
0 (treatment)	-	0.08689		0.07303		0.06931
0.3	0.141	0.09054	-4.2	0.08016	-9.8	0.0724	-4.5
1	0.0562	0.08527	1.9	0.06927	5.1	0.06358	8.3
3	0.0579	0.08752	-0.7	0.07678	-5.1	0.07071	-2
10	0.416	0.07604	12.5	0.06406	12.3	0.0655	5.5
30	3.11	0.05839	32.8	0.0501	31.4	0.04538	34.5
100	4.39	0.05064	41.7	0.04453	39	0.03706	46.5
0	-	0.07876	9.4	0.07553	-3.4	0.07166	-3.4

Inhibition of total cell growth (biomass) and reduction of growth rate for the 0-72h interval were statistically significant at the WAFs prepared at 30 and 100 mg/l, corresponding with measured concentrations of s 3 mg/l (ANOVA-Tukey test of multiple comparison and the Bonferroni t-test, P=0.05).

Validity criteria fulfilled:

yes

Conclusions:

See also attached background material
Nominal concentrations:
According to OECD 201, the factor of the biomass parameter, measured in the blank control between 0 and 72 h, must be at least 16.
With the current test it was found to be 175.0. The test fulfills this validity criterion.
Evaluation of the section-by-section growth rates:
Arithmetic means of the blank control replicates from 0 h to 72 h were: Replicate 1: 1.668; Replicate 2: 1.760; Replicate 3: 1.725; Replicate 4: 1.779; Replicate 5: 1.693; Replicate 6: 1.695. [1/d].
Coefficients of variation in blank control replicates from 0 to 72 h were: Replicate 1: 7.6%; Replicate 2: 10.2%; Replicate 3: 19.5%; Replicate 4: 13.3%; Replicate 5: 1.9%; Replicate 6: 11.7%.
The mean of the replicate coefficients of variation in the section-by-section growth rate was: 10.7%.
According to OECD 201, the mean coefficient of variation, measured in the blank control from 0 to 72 h, must not be higher than 35%. The test fulfills this validity criterion.
The coefficient of variation of the mean specific growth rate replicates in the control between 0 and 72 h was: 5.2%.
According to OECD 201, the coefficient of variation of the mean specific growth rate, measured in the control from 0 to 72 h, must not exceed 7%. The test fulfills this validity criterion.

Measured concentrations:
According to OECD 201, the factor of the biomass parameter, measured in the blank control between 0 and 72 h, must be at least 16.
With the current test it was found to be 175.0. The test fulfills this validity criterion.
Evaluation of the section-by-section growth rates:
Arithmetic means of the blank control replicates from 0 h to 72 h were: Replicate 1: 1.668; Replicate 2: 1.760; Replicate 3: 1.725; Replicate 4: 1.779; Replicate 5: 1.693; Replicate 6: 1.695. [1/d].
Coefficients of variation in blank control replicates from 0 to 72 h were:
Replicate 1: 7.6%; Replicate 2: 10.2%; Replicate 3: 19.5%; Replicate 4: 13.3%; Replicate 5: 1.9%; Replicate 6: 11.7%.
The mean of the replicate coefficients of variation in the section-by-section growth rate was: 10.7%.
According to OECD 201, the mean coefficient of variation, measured in the blank control from 0 to 72 h, must not be higher than 35%. The test fulfills this validity criterion.
The coefficient of variation of the mean specific growth rate replicates in the control between 0 and 72 h was: 5.2%.
According to OECD 201, the coefficient of variation of the mean specific growth rate, measured in the control from 0 to 72 h, must not exceed 7%. The test fulfills this validity criterion.

Description of key information

Slight toxic effects were observed in a test using water accomodated fractions.

Key value for chemical safety assessment

EC50 for freshwater algae:

5.432 mg/L

EC10 or NOEC for freshwater algae:

0.687 mg/L

Additional information

Due to the low water solubility and the impurity profile of the substance a GLP study according to EEC guideline 92/69 (1992) using water accomodated fractions was performed. Algae were exposed to water accommodated fractions at 100, 30, 10, 3, 1, 0.3 mg/l loading after separation of undissolved substance by centrifugation. Due to the high adsorption tendency test vessels were silanised before testing. The EC50 for inhibition of growth corresponds to 100 mg/l loading, the EC50 for biomass reduction corresponds to 16 mg/l loading. The NOEC is at 10 mg/l (loading). Statistical re-evaluation of the data (2017) retrieved following effect values: ErC10 72h 7.8mg/L (loading, WAF), ErC50 72h 103.9 mg/L (loading, WAF).

Since the impurities might have a higher solubility level than the main component, the composition of the test product in the water phase could differ markedly from the original composition of the test product when solutions were prepared at levels above the solubility limit. The concentrations of the impurities in the test solutions were not analysed in the test solutions. Given the observed effects and the number of impurities, the WAF approach was appropriate for this algae study and effects concentrations could be based the loading rates.

Furthermore, concentrations measured in the WAFs prepared at 30 and 100 mg/l exceeded the expected solubility level of 2 mg/l, indicates the presence of an undissolved fraction, in spite the fact that the procedure for preparation of test solutions included centrifuging at a relative high rate to remove any undissolved test substance. This undissolved fraction may have significantly contributed to the effects on algal growth by causing possible mechanical damage.

The study fulfills the validity criteria.

Comments on pH increase:

The pH increase observed in the study is normal in algae studies where significant growth of the algal cells is observed. The guideline states that the pH should not increase by more than 1.5 units in the control group. However, this is not a validity criterion. Algal cells need CO2 from the test system. If there is a strong exponential growth of the cells, the CO2 in the test system will be decreased and as a consequence the pH will increase. Especially, as the test media are not heavily buffered. The present study showed exponential algal cell growth which is clearly indicative of a “healthy” test system. The increasing pH does therefore not affect the outcome of the study. From the comparison of pH and reduction in mean growth it can be seen that only in the lowest concentrations showing no significant effects, pH shifts to pH > 9.0 were observed. Therefore, it can be concluded that the pH drift did not affect the results.