6-(2,4,4-trimethylcyclopentylidene)hexanal

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:

disregarded due to major methodological deficiencies

Study period:

17 May to 07 June 2013

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

This study was performed according to OECD Guideline 201 with GLP certificate. All validity criteria were fulfilled. This study is considered not assignable due to insufficient information provided on the semi-static methodology used. A validation study should be provided to validate this method and, at the time being, a semi-static system is not accepted as an adaptation of the OECD Guideline. With this method, parent and degradation products are present simultaneously, so interactions can occured. In addition, acetone was used as solvent in this study. Because of the potential for interaction with the test chemical resulting in an altered response in the test, solvent use should be restricted to situations where no other acceptable method of test solution preparation is available. The use of solvent is not the best method at the time being. Furthermore, solvents are generally not appropriate for multiconstituent substances, like the test substance (which is a mixture of isomers), where the use of the solvent can preferentially dissolve one or more components and thereby affect the toxicity. Then, the concentration/quantity of solvent used in the treatment solutions was 0.5 mL/L, corresponding to 395 mg/L (with a density of 0.79), which is 5 times higher than the recommended maximum level of solvent (below 0.1 mL/L; OECD No. 23) but is below the NOEC of acetone (which was reported in the ECHA disseminated dossier at 530 mg/L).

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

Dates of inspection: 14 June 2012 / Date of signature: 10 January 2013

Analytical monitoring:

yes

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Chemical name of vehicle: Acetone
- Method: The treatment solutions were prepared in acetone. The different solutions were prepared.
One additional set of vessels were prepared without algae for each test item treatment used for the definitive test. They were maintained under the same environmental conditions as the test system, and were used for analytical check of the test item concentration throughout the test period.
Due to high instability of the test item in water, it was anticipated that the test item treatments will not remain within 80-120 % of the nominal values. As a consequence, the test item treatments were adjusted twice per day. Mean exposure concentrations were further calculated as the geometric mean of the measured concentration in the old and the new media throughout the test period.
- Water control: The controls consisted of 100 mL of mineral medium.
- Solvent control: The solvent controls received the same volume of acetone as used for the test item treatments (0.05 mL).
- Concentration of vehicle in test medium: 0.5 mL/L

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Source (laboratory, culture collection): Strain No. 72 of the Museum National d'Histoire Naturelle (Paris, France) and regularly sub-cultured in the OECD medium at the Phytosafe site.
- Method of cultivation: The inoculum culture was prepared 2-4 days before the start of the test and incubated under the same conditions as the test cultures such to adapt the test algae to test conditions and ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

No data

Test temperature:

21-24 °C

pH:

Start of the test: 7.7-8.8
End of the test: 7.8-8.1

Dissolved oxygen:

No data

Salinity:

No data

Conductivity:

No data

Nominal and measured concentrations:

Nominal concentration: 0.31, 0.61, 1.22, 2.45, 4.89 mg a.i./L
Mean concentrations: 0.1, 0.2, 0.5, 1.1, 2.4 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass Erlenmeyer flasks (250 mL) filled with 100 mL of culture served as test vessels.
- Type: The test vessels were capped with air-permeable stoppers.
- Initial cells density: The initial biomass in the test cultures was the same in all test cultures and sufficiently low to allow exponential growth throughout the incubation period without any risk of nutrient depletion. Historical data at Phytosafe site show that 2 to 5 X 10^3 cells/mL is an appropriate number.
- No. of vessels per concentration in range-finding test: One replicate unit for each the water control and the solvent control, and each of four test item treatments spaced by a ratio of 5.
- No. of vessels per control (replicates): 3 replicate units for the water control.
- No. of vessels per vehicle control (replicates): 3 replicate units for the solvent control and each of five test item treatments in a ratio of 2 and three replicate units for each of the potassium dichromate treatments within which 50 % inhibition should occur.

GROWTH MEDIUM
- Standard medium used: OECD medium (OECD TG 201, according to ISO 8692) was freshly reconstituted by dilution of mineral stock solutions in water.

OTHER TEST CONDITIONS
- The test vessels were kept under orbital shaking so as to keep the algae suspension and to facilitate the transfer of CO2.
- Light intensity and quality: The surface of the cultures received continuous, uniform fluorescent illumination of 60-120 μE/m^-2/S^-1. The light intensity did not deviate by more than 15 % from the average light intensity over the incubation area.

EFFECT PARAMETERS MEASURED
- Algal biomass: The algal biomass in each flask was determined daily over the test period using small volumes removed from the test solution by pipette. These volumes were not replaced. The numeration was done using an electronic cell counter. The results were expressed as cells per liter of solution.
- pH values: The pH of the solutions was measured at the beginning and at end of test. The pH of the control medium was not to increase by more than 1.5 units during the test.

OTHER OBSERVATIONS:
The inoculum culture was examined microscopically to verify that it was normal and healthy in appearance and to detect any abnormal appearance of the algae (as may be caused by exposure to the test substance) at the end of the test.

Reference substance (positive control):

yes

Remarks:

yes, Potassium dichromate at 0.20, 0.60, 0.75 and 1.0 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 2.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 36.7 % inhibiton at 2.4 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield (migrated information)

Remarks on result:

other: 95%-confidence limits = 0.0-65.6 mg/L, Observed interval = 1.1-2.4 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield (migrated information)

Details on results:

Daily numerations:
- The biomass in both the water and the solvent control increased by a factor of approximately 250 and no adverse effects were observed in the solvent control and the test item treatments up to and including 1.0 mg a.i./L. Both specific growth rate and yield were reduced at 5.0 and 25.0 mg a.i./L.

Definitive test:
Specific growth rate:
- The specific growth rate was regular in both the water and the solvent controls throughout the test period. The standard deviation between the three replicate units did not exceed 8 % of the mean value at each sampling time and was less than 1% for the average specific growth rate per day over the entire test period.
- F-variance analysis at a 5 % confidence level was used to judge upon the difference for mean specific growth rate (section by section and total volumes) for each test item treatment compared to the water controls.
- On first day of testing mean specific growth was significantly reduced at 2.4 mg a.i./L but similar to water controls. On second day the growth was considered similar to the controls in every case while on third day it was reduced for the sole 2.4 mg a.i./L test item treatment but similar to water and solvent controls and four lowest test treatment.
- For the entire test period, mean specific growth rates per day was considered as significantly reduced at 0.5, 1.1 and 2.4 mg a.i./L but similar to the water and the solvent controls and for the two lowest test item treatments (0.1 and 0.2 mg a.i./L).
- The regression analysis of the dose response curve was not performed because the highest test item treatment gave only 36.7 % of inhibition (mean value).

Yield:
- Yield in biomass was considered as significantly reduced for the three highest test item concentrations (0.5, 1.1, 2.4 mg a.i./L) but similar to the water controls for the solvent controls and the two lowest test item treatments (0.1 and 0.2 mg a.i./L).
- The regression analysis was performed using the three highest test item treatments. The two lowest test item treatments were excluded due to the plateau phase. Coefficient of determination for the regression analysis was 93.8 %.
- The 95% confidence interval were wide (0.0-65.4 mg a.i./L) because the regression curve was only 94 % confidence and involved only three test item treatments. The preferred observed interval; 1.1-2.4 mg a.i./L.

PH values
- The initial pH values were within the range 7.7-7.8 for the controls and every test item treatment at test initiation.
- At the end of test the pH value of the test media was similar to that of the controls for the three lowest test item treatments. At both 1.1 and 2.4 mg/L reduced the final pH values confirmed that algae growth was inhibited.

Microscopic observations:
- The cells appeared healthy in the controls and the test item treatments. The growth was solely inhibited without any abnormal appearance.

Results with reference substance (positive control):

- ErC50-72h for potassium dichromate calculated as 0.82 mg/L for specific growth rate and 0.34 mg/L for the yield in biomass. The EC50-72 h for potassium dichromate was between 0.6 and 1.0 mg/L for specific growth rate and between 0.2 and 0.75 mg/L for the yield. The results fulfilled the validity criteria based on Phytosafe historical data.

Reported statistics and error estimates:

Statistical determination of the NOEC:
The NOEC was derived from the F-variance analysis at a 5%-confidence level of the response variable (specific growth rate or yield) for each test item treatment compared to the controls. The NOEC corresponds to the highest test item treatment that did not induce significant effects.

EC50 calculations:
Consistently with the classical approach, EC50 values and the related 95% confidence intervals were derived from regression analysis of the concentration response-curves. The regression analysis of the dose response curve was not performed because the highest test item treatment gave only 36.7 % of inhibition (mean value).

Control of test concentrations:

- In first experiment, the test solutions were assessed out from aliquot volumes of test vessels, results showed that recovery of nominal value was less than 86 % in every case. The procedure was reproduced using 10 µL of reconstituted water in closed tubes so as to avoid volatilization of test item, and to ascertain the accuracy of treatment solutions. In this case, recovery of nominal values was within 95-105 % which was taken as a confirmation that test item was readily evaporated from test vessel because of shaking.

- After 5 h of testing, the test item was decreased down to 16-24 % of nominal value and test item was renewed.

- After 24 h, the test item was totally depleted and again treatments were renewed. Successive assessments showed that treatment concentration was decreased to zero with few hours even though it was reproduced twice per day.

Because of high volatility the verification of test item after each medium renewal was performed closed tube instead of aliquots sample of test solution. Results were taken as verification of stability of treatment solution over test period.

Table 6.1.5/1: Definitive test - Mean measured concentrations in the test item treatments and %of the nominal values:

	% of Nominal concentrations mg a.i./L
	0.31	0.61	1.22	2.45	4.89
To	92.8 %	90.4 %	92.5 %	97.8 %	91.1 %
To+7h - Old	31.0 %	25.4 %	29.4 %	26.5 %	29.7 %
To+7h - New	103.6 %	93.6 %	107.1 %	94.6 %	107.0 %
To+22 h - Old	12.7 %	8.8 %	11.9 %	12.8 %	15.9 %
To+22 h - New	94.5 %	93.4 %	92.8 %	104.9 %	99.9 %
To+31h - Old	27.1 %	20.1 %	24.7 %	28.6 %	31.2 %
To+31h - New	120.2 %	113.4 %	114.1 %	119.0 %	139.3 %
To+46h - Old	10.2 %	9.0 %	11.1 %	15.0 %	17.4 %
To+46h - New	91.4 %	87.9 %	109.4 %	98.3 %	92.6 %
To+55h - Old	21.8 %	16.9 %	22.9 %	22.3 %	27.3 %
To+55h - New	94.7 %	95.0 %	94.5 %	94.6 %	97.0 %
To+72h	19.7 %	14.8 %	23.1 %	22.5 %	26.4 %
Geometric mean	0.1 mg/L	0.2 mg/L	0.5 mg/L	1.1 mg/L	2.4 mg/L

Table 6.1.5/2: Definitive test - Mean measured specific growth rates per day (section-by-section and total)

	0 to 24 h	24 to 48 h	48 to 72 h	Total period
Water control Mean ± S.D.	1.89 ± 0.04	1.94 ± 0.06	1.66 ± 0.06	1.83 ± 0.02
Solvent control Mean ± S.D.	1.89 ± 0.09	1.93 ± 0.08	1.61 ± 0.05	1.81 ± 0.01
Test item 0.1 mg/L- Mean ± S.D.	1.92 ± 0.02	1.92 ± 0.08	1.68 ± 0.03	1.84 ± 0.01
Test item 0.2 mg/L ± Maen S.D.	1.85 ± 0.10	1.91 ± 0.01	1.64 ± 0.05	1.80 ± 0.03
Test item 0.5 mg/L Mean ± S.D.	1.87 ± 0.08	1.83 ± 0.04	1.67 ± 0.05	1.79 ± 0.02
Test item 1.1 mg/L Mean ± S.D.	1.74 ± 0.09	1.76 ± 0.16	1.63 ± 0.05	1.71 ± 0.01
Test item 2.4 mg/L Mean ± S.D.	1.34 ± 0.10	1.78 ± 0.09	0.35 ± 0.13	1.16 ± 0.05

Table 6.1.5/3: F-variance analysis for mean specific growth rate per day in the test item treatments compared to controls (Threshold value for F = 7.71 mg/L)

	Solvent control	Marinal
		0.1 mg/L	0.2 mg/L	0.5 mg/L	1.1 mg/L	2.4 mg/L
0 to 24 h	F = 0.01	F = 1.13	F = 0.59	F = 0.29	F = 7.45	F = 80.64
24 to 48 h	F = 0.04	F = 0.23	F = 0.67	F = 7.35	F = 3.55	F = 6.85
48 to 72 h	F = 1.21	F = 0.18	F = 0.23	F = 0.05	F = 0.55	F = 250.96
Total period	F = 2.89	F = 0.30	F = 2.20	F = 10.09	F= 113.69	F = 429.74

Table 6.1.5/4: Definitive test – Percentage inhibition of specific growth rates per day (section-by-section

	0 to 24h	24 to 48h	48 to 78h	Total period
Solvent control	0.3 %	0.6 %	3.0 %	1.2 %
Marinal
0.1 mg a.i./L	-1.5 %	1.4 %	-1.0 %	-0.4 %
0.2 mg a.i./L	2.5 %	1.5 %	1.3 %	1.8 %
0.5 mg a.i./L	1.4 %	5.9 %	-0.6 %	2.4 %
1.1 mg a.i./L	8.3 %	9.3 %	2.0 %	6.7 %
2.4 mg a.i./L	29.0 %	8.2 %	78.8 %	36.7 %

(1)  Negative values indicate the growth rate was increased as compared to controls.

Table 6.1.5/4: Definitive test - Yield of biomass throughout the 72-hour incubation period with statistical analysis of main yield in mean and test item treatments:

Groups	Yield	Statistical analysis (Threshold value for F = 7.71)
Water control	9.06E+08 (4.83E+07)	-
Solvent control	8.47E+08 (3.73E+07)	F = 2.83
Test item Mean ± S.D.
0.1 mg a.i./L	9.24E+08 (3.04E+07)	F = 0.28
0.2 mg a.i./L	8.24E+08 (8.17E+07)	F = 2.27
0.5 mg a.i./L	7.95E+08 (3.88E+07)	F = 9.77
1.1 mg a.i./L	6.24E+08 (1.86E+07)	F = 89.29
2.4 mg a.i./L	1.18E+08 (1.86E+07)	F = 696.43

VALIDITY OF THE TEST RESULTS

The test was considered as valid in the light of the following criteria:

- The biomass in the control cultures increased exponentially by a factor of at least 16 over the 72-hour test period.

- The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35 % (this criterion applies to the mean value of the coefficients of variation calculated for replicate control cultures).

- The coefficient of variation for average specific growth rates over the entire test period in replicate control cultures did not exceed 7 %.

Additionally, the EC50-72 h for potassium dichromate was between 0.6 and 1.0 mg/L for the specific growth rate and between 0.2 and 0.75 mg/L for the yield. The results fulfilled the validity criteria based on Phytosafe historical data.

- Due to high volatility of the test item, the test item treatments were not maintained within 80-120 % of the nominal value during the test period even though the test item treatments were set at 120 % of the nominal values and the treatments were adjusted twice per day. The calculations were based on the geometric mean of the measured concentration during the test period in the old and the new media.

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, the ErC50 (growth rate) was > 2.4 mg/L (36.7 % inhibition at 2.4 mg/L) and EyC50 (yield) was 1.2 mg/L (95% confidence interval = 0.0-65.6 mg/L) of the test item to Desmodesmus subspicatus. The NOEC was 0.2 mg/L based on specific growth rate (36.7 % inhibition at 2.4 mg/L) and 0.2 based on yield.

Executive summary:

In an algal growth inhibition study performed according to OECD Guideline 201 and in compliance with GLP, the freshwater green algae species Desmodesmus subspicatus was first exposed to test item for 72 h, under semi-static conditions and constant illumination, shaking at a temperature of 21-24 °C. A range-finding study was performed using one replicate for control and each of the five test item treatments between 0.04 and 25 mg/L and definitive test was performed using three replicate units for each of five test item treatments and for controls. The percent inhibition for growth rate and yield was determined.

No adverse effects were observed in the solvent control and the test item treatments up to and including 1.0 mg a.i./L in range finding study. Both specific growth rate and yield were reduced at 5.0 and 25.0 mg a.i./L while in definitive the specific growth rate was regular in both water and solvent controls throughout the test period and the standard deviation between the three replicate units did not exceed 8 % of the mean value at each sampling time and was less than 1% for the average specific growth rate per day over the entire test period. For entire period mean specific growth rates per day was considered as significantly reduced at 0.5, 1.1 and 2.4 mg a.i./L but similar to the water controls for the solvent controls and for the two lowest test item treatments (0.1 and 0.2 mg a.i./L). Regression analysis of the dose response curve was not performed as the highest test item treatment gave only 36.7 % of inhibition (mean value).

Yield in biomass was considered as significantly reduced for three highest test item concentrations (0.5, 1.1, 2.4 mg a.i./L) but similar to the water control, solvent controls and the two lowest test item treatments (0.1 and 0.2 mg a.i./L). The regression analysis was performed using the three highest test item treatments. The two lowest test item treatments were excluded due to the plateau phase. The 95% confidence interval were wide (0.0-65.4 mg a.i./L) because the regression curve was only 94 % confidence and involved only three test item treatments.

Under the test conditions, the ErC50 (growth rate) was > 2.4 mg/L (36.7 % inhibition at 2.4 mg/L) and EyC50 (yield) was 1.2 mg/L (95% confidence interval = 0.0-65.6) of the test item to Desmodesmus subspicatus. The NOEC was 0.2 mg/L based on specific growth rate and yield.

All validity criteria were fulfilled. However, this study is considered not assignable due to insufficient information provided on the semi-static methodology used. A validation study should be provided to validate this method and, at the time being, a semi-static system is not accepted as an adaptation of the OECD Guideline. With this method, parent and degradation products are present simultaneously, so interactions can occured. In addition, acetone was used as solvent in this study. Because of the potential for interaction with the test chemical resulting in an altered response in the test, solvent use should be restricted to situations where no other acceptable method of test solution preparation is available. The use of solvent is not the best method at the time being. Furthermore, solvents are generally not appropriate for multiconstituent substances, like the test substance (which is a mixture of isomers), where the use of the solvent can preferentially dissolve one or more components and thereby affect the toxicity. Then, the concentration/quantity of solvent used in the treatment solutions was 0.5 mL/L, corresponding to 395 mg/L (with a density of 0.79), which is 5 times higher than the recommended maximum level of solvent (below 0.1 mL/L; OECD No. 23) but is below the NOEC of acetone (which was reported in the ECHA disseminated dossier at 530 mg/L).

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 28 to November 4, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD Guideline 201 without restrictions.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Issue on 17 March 2021

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Appearance: Light yellow translucent liquid
- Source and lot/batch number of test material: V. MANE FILS / 0000634308
- Expiration date of the lot/batch: March 2022
- Purity test date: April 14, 2021

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature. Store in a cool, dry place, protected from light.

Analytical monitoring:

yes

Details on sampling:

Single samples for analysis were taken from the control and all test solutions (from a replicate of each test concentration with algae dedicated exclusively to chemical analysis) at the start and every day thereafter until the end of the test.

Vehicle:

yes

Remarks:

test water

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The mixing vessel was a cylindrical glass bottle sealed with a screw cap and fitted with a drain port near the bottom for drawing off the saturated stock solution. The volume of the mixing vessel was approximately 2 L. A magnetic stirring bar was placed in the vessel and test water was added. Then 200.3 mg test item were weighed on a weighing boat that afterwards was placed above the mixing vessel and rinsed with test water. The mixing vessel was then carefully filled with the remaining volume of test water to obtain 2 L of stock solution and thereafter was closed immediately. Mixing was initiated with the vortex in the centre extending maximally to around 10% of the vessel depth from the top to the bottom of the vessel. After approx. 21.5 hours of gentle stirring in the dark at room temperature, the contents of the vessel were allowed to stand undisturbed for at least 1 hour before use. The first 100 mL were discarded via the drain port. Samples were taken from the remaining stock solution and chemically analysed. At this time, the concentration of the stock solution was lower than expected (2.57 mg/L, instead of the 4.14 mg/L obtained during the range-finding test in a 1-L mixing vessel), which is why it was decided to extend the stirring period by a further 3 to 4 hours in an attempt to achieve a higher saturated concentration. Then the stock solution was diluted with test water as necessary, based on the measured concentration of the stock solution (= 2.81 mg/L), and fixed amount of inoculum in order to obtain the required test concentrations in the test vessels and a cell density of 5 x 10^3 cells/mL/vessel. At the start of the test, the test solutions in all test vessels were observed to be clear and colourless. The Tyndall effect (checked via laser beam) was negative in all treatments. The pH of the test solutions was not adjusted.
- Controls: Test water without test substance but treated in the same way as the test substance solutions.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Museum National d’Histoire Naturelle - 12, rue Buffon, Case N°19 -75005 PARIS, bred in the Laboratoires des Pyrénées et des Landes under standardised conditions, according to the test guidelines.
- Stock culture Algae stock cultures were started by inoculating growth medium (= test water) 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 regime: Continuous illumination
- Light intensity: 4440-8880 lux
- Pre-culture: 4 days before the start of the test, cells from the algal stock culture were inoculated in test water at a cell density of 1 x 10^4 cells/mL. The preculture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Test type:

static

Water media type:

freshwater

Remarks:

Original medium from OECD TG 201

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.0 and 23.7 °C throughout the test (average value: 23.3 °C)

pH:

0 h: 8.25 to 8.32
and at 72 h: 8.56 to 9.71

Nominal and measured concentrations:

Nominal concentrations: 0.2, 0.4, 0.7, 1.3 and 2.5 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass closed flasks with ground glass stopper, completely filled with test solution with minimum headspace.
- Type: closed
- Initial cells density: An initial cell density of 5 x 10^3 cells/L using the exponentially growing pre-culture.
- No. of vessels per concentration (replicates): 3 replicates of each test concentration
- No. of vessels per control (replicates): 6 controls
- Moreover, additional replicates of each treatment with algae were prepared for chemical analysis (destructive samples) in order to determine maintenance of actual concentrations.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Test water (Original medium of OECD TG 201)

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: The test was carried out without adjustment of the pH. pH Should not vary by more than 1.5 units at the end of the test in the control.
- Test environment: Controlled environment cabinet (23°C ± 2 °C); vessels were distributed randomly in the incubator and redistributed over the test at t=24h and t=48h. During incubation, the algal cells were kept in suspension by continuous magnetic stirring (250 rpm).
- Light intensity and quality: Continuous illumination with a light intensity of 4440-8880 lux and did not vary more than ± 15% from the average light intensity over the incubation area.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Cell numbers were counted daily by microscope using a counting chamber.
- pH: At the start and the end of the test in one vessel per concentration and the control (same vessel at t=0h and t=72h).
- Temperature of medium: Measured continuously in the growth chamber, over the study period.
- Light intensity: Light intensity was measured once (t=0h) during the test at 5 positions distributed over the experimental area at the surface of the test media.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.88

Range finding study
- Test concentrations: 0.05, 0.20, 0.50, 1.00 and 3.73 mg/L
- Results used to determine the conditions for the definitive study: Percentage inhibition of algal growth rate caused by the test item at 72 h were 2.6, -0.4, 2.8, 8.9 and 73.7% at 0.05, 0.20, 0.50, 1.00 and 3.73 mg/L, respectively. Based on the results of a range-finding test and on the concentration of the stock solution of the test item, test solutions used in the definitive test were prepared to obtain the following nominal concentrations (spaced by a factor of approximately 1.88): 0.2, 0.4, 0.7, 1.3 and 2.5 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.369 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% cl 0.304 – 0.430 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.115 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% cl 0.065 – 0.159 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.551 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% cl 0.478 – 0.620 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.179 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% cl 0.120 – 0.228 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.184 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% cl 1.069 – 1.324 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.419 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% cl 0.351 – 0.504 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

The 72-hour EC50 for the parameters growth rate and yield were determined to be 1.184 mg/L and 0.419 mg/L, respectively. The 72-hour EC10 for growth rate was 0.369 mg/L and for yield was 0.115 mg/L. The 72-hour NOEC for growth rate was 0.140 mg/L and for yield was less than 0.140 mg/L. Since analytical determinations were performed at t=48 h, the endpoint values at t=48 h were also determined (validity criteria of the study at t=48h were respected).
The 48-hour EC50 for the parameters growth rate and yield were determined to be 1.766 mg/L and 0.542 mg/L, respectively. The 48-hour EC10 for growth rate was 0.406 mg/L and for yield was 0.098 mg/L. The 48-hour NOEC value for both growth rate and yield was 0.170 mg/L.

Results with reference substance (positive control):

- Results with reference substance valid: Yes
- The 72h-EC50 was 0.759 mg/L for the parameter growth rate. Hence, the sensitivity of this batch of Pseudokirchneriella subcapitata was consistent with the level proposed by the ISO 8692 (expected 72h-ErC50: 0.65 mg/L1 to 1.73 mg/L).

Table 6.1.5/1: Mean specific growth rate in P. subcapitata during the final test

 

t	Replicate	Nominal concentration and corresponding analytically confirmed concentration (geometric mean) after 48h and after 72h (mg test item/L)
		Control (0) (0)	0.2 (0.17) (0.14)	0.4 (0.34) (0.25)	0.7 (0.65) (0.46)	1.3 (1.06) (0.69)	2.5 (2.39) (2.16)
t=0 to 24 h	1	2.079	1.569	1.163	0.875	0.470	0.470
	2	1.856	2.079	1.569	2.175	1.569	0.875
	3	1.723	1.723	1.386	1.569	1.723	0.875
	4	2.175	-	-	-	-	-
	5	1.856	-	-	-	-	-
	6	1.856	-	-	-	-	-
	Mean	1.924	1.790	1.373	1.540	1.254	0.740
	SD	0.1681	0.2620	0.2031	0.6501	0.6831	0.2341
	% Red.	-	7.0	28.7	20.0	34.8	61.5
t=0 to 48 h	1	1.844	1.694	1.694	1.707	1.275	0.784
	2	2.005	1.910	1.666	1.694	1.386	0.582
	3	2.005	1.769	1.694	1.456	1.334	0.784
	4	1.781	-	-	-	-	-
	5	1.944	-	-	-	-	-
	6	1.936	-	-	-	-	-
	Mean	1.919	1.791	1.685	1.619	1.332	0.717
	SD	0.0901	0.1097	0.0160	0.1412	0.0558	0.1170
	% Red.	-	6.7	12.2	15.6	30.6	62.7
t=0 to 72 h	1	1.596	1.524	1.521	1.422	1.155	0.388
	2	1.660	1.558	1.386	1.471	1.172	0.523
	3	1.613	1.573	1.504	1.382	1.147	0.292
	4	1.543	-	-	-	-	-
	5	1.632	-	-	-	-	-
	6	1.617	-	-	-	-	-
	Mean	1.610	1.552	1.471	1.425	1.158	0.401
	SD	0.0394	0.0249	0.0736	0.0443	0.0126	0.1161
	% Red.	-	3.6	8.7	11.5	28.1	75

Values were extracted from the computer program ToxRat

% Inh.: %Inhibition of growth rate relative to the control determined by ToxRat

 

Table 6.1.5/2: Yield of algal cells during the final test

 

t	Replicate	Nominal concentration and corresponding analytically confirmed concentration (geometric mean) after 48h and after 72h (mg test item/L)
		Control (0) (0)	0.2 (0.17) (0.14)	0.4 (0.34) (0.25)	0.7 (0.65) (0.46)	1.3 (1.06) (0.69)	2.5 (2.39) (2.16)
t=24 h	1	3.5	1.9	1.1	0.7	0.3	0.3
	2	2.7	3.5	1.9	3.9	1.9	0.7
	3	2.3	2.3	1.5	1.9	2.3	0.7
	4	3.9	-	-	-	-	-
	5	2.7	-	-	-	-	-
	6	2.7	-	-	-	-	-
	Mean	3.0	2.6	1.5	2.2	1.5	0.6
	SD	0.60	0.83	0.40	1.62	1.06	0.23
	% Red.	-	13.5	49.4	27.0	49.4	80.9
t=48 h	1	19.5	14.3	14.3	14.7	5.9	1.9
	2	27.1	22.3	13.5	14.3	7.5	1.1
	3	27.1	16.7	14.3	8.7	6.7	1.9
	4	17.1	-	-	-	-	-
	5	23.9	-	-	-	-	-
	6	23.5	-	-	-	-	-
	Mean	23.0	17.8	14.0	12.6	6.7	1.6
	SD	4.04	4.11	0.46	3.35	0.80	0.46
	% Red.	-	22.9	39.1	45.4	70.9	92.9
t=72 h	1	59.5	47.9	47.5	35.1	15.5	1.1
	2	72.3	53.1	31.5	40.7	16.3	1.9
	3	62.7	55.5	45.1	31.1	15.1	0.7
	4	50.7	-	-	-	-	-
	5	66.3	-	-	-	-	-
	6	63.5	-	-	-	-	-
	Mean	62.5	52.2	41.4	35.6	15.6	1.2
	SD	7.21	3.89	8.63	4.82	0.61	0.61
	% Red.	-	16.5	33.8	43.0	75.0	98.0

Values were extracted from the computer program ToxRat

% Red.: %Reduction in yield relative to the control determined by ToxRat

 

Table 6.1.5/3: ErCx, EyCx, NOErC and NOEyC values after 48 and 72 hours

 

Parameter	Growth rate in mg/L (and 95% conf. limits)	Yield in mg/L (and 95% conf. limits)
48-hour EC10	0.406 (0.283 – 0.517)	0.098 (0.038 – 0.161)
48-hour EC20	0.672 (0.531 – 0.798)	0.177 (0.090 – 0.256)
48-hour EC50	1.766 (1.524 – 2.127)	0.542 (0.413 – 0.703)
48-hour NOEC	0.170	0.170
72-hour EC10	0.369 (0.304 – 0.430)	0.115 (0.065 – 0.159)
72-hour EC20	0.551 (0.478 – 0.620)	0.179 (0.120 – 0.228)
72-hour EC50	1.184 (1.069 – 1.324)	0.419 (0.351 – 0.504)
72-hour NOEC	0.140	<0.140

 

Physico-chemical parameter values throughout the test

The temperature in the incubator was situated between 22.0 and 23.7 °C throughout the test (average value: 23.3 °C). The mean light intensity was 4906 lux (range: 4779-5083 lux). Thus, all test conditions remained within the ranges prescribed by the study plan (pH: not varying by more than 1.5 units at the end of the test in the control; temperature: 23 ± 2 °C, constant within 2 °C; light intensity: 4440-8880 lux and not varying more than ± 15% from the average light intensity over the incubation area).

 

Analytical results

Samples taken from the control and all test concentrations were analysed at the start and every day thereafter until the end of the test in order to determine actual concentrations of the test item.

Chemical analysis revealed unavoidable test item losses from t=24 h until the end of the test, despite every effort made to maintain the concentrations of the test item (closed conditions with minimum headspace). These decreases were lower at the highest (and toxic) test concentration, suggesting potential adsorption/metabolism effects by algae at lower (less or non-toxic) concentrations. Moreover, analytical results showed the appearance of a new chromatographic peak in all 48 h and 72 h-old solutions, suggesting the likely formation of a degradation product. Since the deviation of the exposure concentrations was greater than ± 20% of the initial concentrations, the evaluation of the effects on P. subcapitata was based on the geometric means of the analytically confirmed concentrations after 48 and 72 h.

 

Validity criteria of the study

Cell density in controls 126 fold increase within 72 hours (and 47.1-fold increase within 48 hours).

Coefficient of variation:

1. The mean coefficient of variation for section-by-section specific growth rates in the control cultures was 12.0% in 72 hours (and 15.3% within 48 hours).

2. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 2.4% (and 4.7% within 48 hours).

Thus the validity criteria were respected in this study.

Validity criteria fulfilled:

yes

Conclusions:

Under the experimental conditions and based upon analytically confirmed concentrations, the 72-hour EC50 for the parameters growth rate and yield were determined to be 1.184 mg/L and 0.419 mg/L, respectively. The 72-hour EC10 for growth rate was 0.369 mg/L and for yield was 0.115 mg/L. The 72-hour NOEC for growth rate was 0.140 mg/L and for yield was less than 0.140 mg/L.

Executive summary:

In an algal growth inhibition study performed according to OECD Guideline 201 and in compliance with GLP, freshwater green algae species Pseudokirchneriella subcapitata was exposed to test item in a closed static test at the nominal concentrations of 0.2, 0.4, 0.7, 1.3 and 2.5 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 22.0-23.7 °C. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. Samples taken from the control and all test concentrations were analysed at the start and every day thereafter until the end of the test in order to determine if concentrations of the test item were maintained.

 

Specific analysis of samples taken from all test concentrations revealed unavoidable losses of test item. These decreases were lower at the highest (and toxic) test concentration, suggesting potential adsorption/metabolism effects by algae at lower (less toxic) concentrations. Since the deviation of the exposure concentrations was greater than ± 20% of the initial concentrations, the results were based on the geometric means of the analytically confirmed concentrations.

 

Under the experimental conditions and based upon analytically confirmed concentrations, the 72-hour EC50 for the parameters growth rate and yield were determined to be 1.184 mg/L and 0.419 mg/L, respectively. The 72-hour EC10 for growth rate was 0.369 mg/L and for yield was 0.115 mg/L. The 72-hour NOEC for growth rate was 0.140 mg/L and for yield was less than 0.140 mg/L. Since analytical determinations were performed at t=48 h, the endpoint values at t=48 h were also determined (validity criteria of the study at t=48h were respected). The 48-hour EC50 for the parameters growth rate and yield were determined to be 1.766 mg/L and 0.542 mg/L, respectively. The 48-hour EC10 for growth rate was 0.406 mg/L and for yield was 0.098 mg/L. The 48-hour NOEC value for both growth rate and yield was 0.170 mg/L.

Description of key information

OECD 201, GLP, Key study, K1 : 

72h-ErC50 = 1.18 mg/L (95% CL: 1.07 - 1.32 mg/L)

72h-NOECr = 0.140 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

1.18 mg/L

EC10 or NOEC for freshwater algae:

0.14 mg/L

Additional information

Two experimental study are available to assess the toxicity of the registered substance to aquatic algae.

The first data (LPL, 2021) is an experimental key study. In an algal growth inhibition study performed according to OECD Guideline 201 and in compliance with GLP, the freshwater green algae species Pseudokirchneriella subcapitata was exposed to test item in a closed static test at the nominal concentrations of 0.2, 0.4, 0.7, 1.3 and 2.5 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 22.0-23.7 °C. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. Samples taken from the control and all test concentrations were analysed at the start and every day thereafter until the end of the test in order to determine if concentrations of the test item were maintained.

 Specific analysis of samples taken from all test concentrations revealed unavoidable losses of test item. These decreases were lower at the highest (and toxic) test concentration, suggesting potential adsorption/metabolism effects by algae at lower (less toxic) concentrations. Since the deviation of the exposure concentrations was greater than ± 20% of the initial concentrations, the results were based on the geometric means of the analytically confirmed concentrations.

 

Under the experimental conditions and based upon analytically confirmed concentrations, the 72-hour EC50 for the parameters growth rate and yield were determined to be 1.184 mg/L and 0.419 mg/L, respectively. The 72-hour EC10 for growth rate was 0.369 mg/L and for yield was 0.115 mg/L. The 72-hour NOEC for growth rate was 0.140 mg/L and for yield was less than 0.140 mg/L. 

The second data (phytosafe, 2013)  is an experimental study and was disregarded due to major methodological deficiencies. In an algal growth inhibition study performed according to OECD Guideline 201 and in compliance with GLP, the freshwater green algae species Desmodesmus subspicatus was first exposed to test item for 72 h, under semi-static conditions and constant illumination, shaking at a temperature of 21-24 °C. 

Under the test conditions, the ErC50 (growth rate) was >2.4 mg/L of the test item to Desmodesmus subspicatus. The NOEC was 0.2 mg/L based on nominal concentrations.

 

All validity criteria were fulfilled. This study is considered not assignable due to insufficient information provided on the semi-static methodology used. A validation study should be provided to validate this method and, at the time being, a semi-static system is not accepted as an adaptation of the OECD Guideline. With this method, parent and degradation products are present simultaneously, so interactions can occur. In addition, acetone was used as solvent in this study. Because of the potential for interaction with the test chemical resulting in an altered response in the test, solvent use should be restricted to situations where no other acceptable method of test solution preparation is available. The use of solvent is not the best method at the time being. Furthermore, solvents are generally not appropriate for multiconstituent substances, like the test substance (which is a mixture of isomers), where the use of the solvent can preferentially dissolve one or more components and thereby affect the toxicity. Then, the concentration/quantity of solvent used in the treatment solutions was 0.5 mL/L, corresponding to 395 mg/L (with a density of 0.79), which is 5 times higher than the recommended maximum level of solvent (below 0.1 mL/L; OECD No. 23) but is below the NOEC of acetone (which was reported in the ECHA disseminated dossier at 530 mg/L).