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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 17 September 2018 to 19 March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspection dates 29-30-31 May 2018 / Signature date 15 November 2018

Specific details on test material used for the study:

- Storage conditions: To be stored refrigerated (2-8°C) protected from light, stored under nitrogen after first opening.
- Vapour pressure: 1.3 Pa at 20°C and 2.4 Pa at 25°C (OECD Guideline 104, effusion method, LPL 2018)
- Water solubility: 39.7 mg/L (iSafeRat v1.7 prediction). Experimental study on-going at the date of the present study
- Log Kow: 3.93 (iSafeRat v1.7 prediction). Experimental study on-going at the date of the present study
- Stability: Stable (less than 10% loss) from pH 2-12 (Firmenich internal data)

Analytical monitoring:

yes

Details on sampling:

Single samples for analysis were taken from the control and all test concentrations (from a replicate of each test concentration with algae dedicated exclusively to chemical analyses) at the start
and the end of the test.
Test samples (specimen) were injected directly after a dilution by a factor of two with acetonitrile (500µL sample + 500 µL acetonitrile). If the sample concentrations were too high and not included in the concentration range of the calibration, the samples were diluted appropriately with a test water/acetonitrile (50/50 v/v) mixture.
Samples were injected as soon as possible after their preparation.
A blank sample of test water/acetonitrile (50/50) was injected every analytical sequence in order to check that no instrument or glassware contamination had occurred. Blank samples were considered as valid if their concentrations were below the limit of detection.

Vehicle:

no

Details on test solutions:

PREPARATION OF STOCK AND TEST SOLUTIONS
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 1 L. A magnetic stirring bar was placed in the vessel and approx. 1 L test water was added. Then an excess of the test item (approx. 1000 mg) was carefully added directly to the surface of the test water. The mixing vessel was thereafter closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 23.5 hours of gentle stirring in the dark at room temperature, the contents of the vessel was 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. Then the stock solution was diluted with test water as necessary based on the measured concentration of the stock solution (≈ 38.7 mg/L) and a fixed amount of inoculum to obtain the required test concentrations in the test vessels and a cell density of 5.103 cells/mL per vessel. At the start of the test, the test solutions in test vessels were observed to be clear and colourless.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- 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.
- Method of cultivation: 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. 2 to 4 days before the start of the test, cells from the algal stock culture were inoculated in test water at a cell density of 1.104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

The temperature in the incubator was situated between 21.9 and 23.7°C throughout the test (average value: 23.5°C), and complied with the requirements as laid down in the study plan (23°C ±
2°C, constant within 2°C).

pH:

From 8.24 to 9.94

Dissolved oxygen:

No data

Salinity:

Not applicable

Conductivity:

No data

Nominal and measured concentrations:

- Nominal concentrations: 0.5, 1.0, 2.0, 4.0 and 8.0 mg/L.
- Geometric mean measured concentrations: 0.81, 1.01, 2.08, 4.44 and 8.75 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 (delete if not applicable): closed
- Initial cells density: An initial cell density of 5.103 cells/mL using the exponentially growing pre-culture.
- No. of vessels per concentration (replicates): 3 replicates
- No. of vessels per control (replicates): 6 replicates. Test water without test substance but treated in the same way as the test substance solutions.
- No. of vessels per vehicle control (replicates): not applicable
Moreover, a replicate of each treatment with algae was prepared for chemical analyses.

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
Original medium from OECD TG 201
Since the test was performed in sealed conditions, additional sodium bicarbonate was added to test water to insure a satisfactory CO2 supply for the algal growth (for all treatments and inoculum suspension): 7 mL of NaHCO3 was added to the sterilised water during test water reconstitution (instead of 1 mL) to obtain a final concentration of 350 mg/L, according to the study plan.

OTHER TEST CONDITIONS
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.
Continuous illumination with a light intensity of 4,440-8,880 lux and did not vary more than ± 15% from the average light intensity over the incubation area.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell numbers were counted daily by microscope using a counting chamber.
- Chlorophyll measurement: no
- Other: Absence of undissolved test item in the stock solution was checked by determination of turbidity (ISO 7027, Water quality - Determination of turbidity). Turbidity analysis will not be performed in compliance with the OECD GLP principles but in accordance with ISO 17025.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Range finding study: In the range-finding test, algal cells were exposed to a series of nominal test concentrations of 0.5, 1.0, 10.0, 32.0 mg/L and to a concentration close to the solubility limit of the test item in test water (≈ 37.5 mg/L), and to a control. The test system was maintained in a temperature controlled cabinet (23°C ± 2°C) with continuous illumination for a period of 72 hours. The geometric mean measured concentrations were determined to be 1.14, 9.76, 32.96 and 42.65 mg/L. The test concentration of 0.5 mg/L (nominal) was not analysed.
- Results used to determine the conditions for the definitive study: The percentage inhibition of growth rate relative to the control after 72 hours were 2.9%, 9.7%, 95.6%, 99.9% and 99.9% at 0.5 (nominal), 1.14, 9.76, 32.96 and 42.65 mg/L, respectively, based on measured concentrations.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (Batch n° 16H244108; purity Min. 99.8%; expiry date 16 January 2019)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.845 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 4.329-5.432 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.256 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 1.523-2.781 mg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

5.369 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 4.716-6.030 mg/L

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

2.058 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 1.364-2.631 mg/L

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.01 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

PHYSICO-CHEMICAL PARAMETER VALUES THROUGHOUT THE TEST
Although the pH level in the control varied by more than 1.5 units at the end of the test (1.57 units of difference) this was not considered to have affected the integrity of the study. The cause of the pH increases in the controls and test concentrations was certainly due to the substantial algal growth in conjunction with closed conditions used in the test, despite the additional sodium bicarbonate.
The mean light intensity was of 5592 lux (range: 5358-5938 lux), which remained within the ranges prescribed by the study plan (range: 4440-8880 lux and shall not vary more than ± 15% from
the average light intensity over the incubation area).
The temperature in the incubator was situated between 21.9 and 23.7°C throughout the test (average value: 23.5°C), and complied with the requirements as laid down in the study plan (23°C ±
2°C, constant within 2°C).
Furthermore, turbidity measurements revealed the absence of undissolved material in the stock solution (represented by turbidity analysis of the stock solution, in comparison with the turbidity value
of the control).

ANALYTICAL RESULTS
The analytical results of this test showed that concentrations of test solutions prepared following the outlined procedures were overall stable. Indeed, analyses of samples taken from all test concentrations at the start and the end of the test revealed that test item levels were stable, with losses of test item < 20% compared to initial values. Furthermore, test item concentrations were satisfactorily maintained within ± 20% of the nominal concentrations throughout the test, except for the geometric mean of the measured concentrations at 0.5 mg/L which was higher than the corresponding nominal concentration over the study period (162%); no explanation for this difference was found. Therefore, the evaluation of the effects on P. subcapitata was based on the
geometric means of the measured concentrations.
See table 6.1.5/3 in "Any other information on results incl. tables"

BIOLOGICAL RESULTS
See tables 6.1.5/1-2 in "Any other information on results incl. tables"
Under the experimental conditions and based on the geometric means of measured exposure concentrations, the 72h-ErC50 and 72h-ErC10 values for the parameter growth rate were determined to be 4.845 mg/L and 2.256 mg/L, respectively. Since the 72h-NOEC value for growth rate could not be determined, the endpoint values at t=48h were also determined in order to have a complete set NOEC/EC10/EC50. Indeed, according to OECD 201 section 11, it is possible to shorten the test period to 48 hours whenever unlimited, exponential growth during the test has been obtained. This corresponds to at least a 16-fold growth in controls. Since the cell density in the control cultures increased by a factor of 38 within 48 hours exposure period, the results can also be provided after 48h according to OECD 201. The 48d-ErC50 and 48h-ErC10 values for the parameter growth rate were determined to be 5.369 mg/L and 2.058 mg/L, respectively. The 48h-NOEC value for growth rate was 1.010 mg/L.

Results with reference substance (positive control):

On September 11, 2018 (KA18-002; most recent test), the 72h-EC50 was 1.116 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-EC50: 0.92 mg/L to 1.46 mg/L).

Reported statistics and error estimates:

The evaluation of the effects was based on measured concentrations. The software ToxRat® Professional was used to perform statistical analyses. Complementary statistical analyses were performed at t=72h using another statistical spreadsheet (using Excel®) for the validity criteria of the study.

Table 6.1.5/1: Algae cell densities during the final test (expressed as density of algal cells/mL x 10^4)

	Replicate	Nominal concentration and corresponding measured concentration (geometric mean)  (mg test item.L^-1)
		Control (0)	0.5 (0.81)	1.0 (1.01)	2.0 (2.08)	4.0 (4.44)	8.0 (8.75)
t=24h	1	3.2	2.8	3.2	2.8	1.6	1.2
	2	4.0	4.0	4.0	4.4	0.8	0.4
	3	6.0	3.6	3.6	3.2	2.8	0.0
	4	6.0
	5	2.8
	6	4.4
	Mean	4.4	3.5	3.6	3.5	1.7	0.5
	Std. Dev.	1.36	0.61	0.40	0.83	1.01	0.61
t=48h	1	20.0	15.2	16.4	16.0	6.4	1.6
	2	17.6	19.6	16.0	11.2	4.0	0.8
	3	22.4	17.6	17.6	9.2	5.6	0.4
	4	20.8
	5	16.0
	6	18.0
	Mean	19.1	17.5	16.7	12.1	5.3	0.9
	Std. Dev.	2.35	2.20	0.83	3.49	1.22	0.61
t=72h	1	39.2	30.8	37.6	38.0	10.8	0.4
	2	52.0	30.0	36.8	26.4	6.8	0.4
	3	58.4	32.4	28.0	26.4	8.4	0.0
	4	61.2
	5	48.0
	6	54.8
	Mean	52.3	31.1	34.1	30.3	8.7	0.3
	Std. Dev.	7.91	1.22	5.33	6.70	2.01	0.23

At test start 5000algal cells.mL^-1were incubated; 6 replicates of the controls and 3 replicates of each test concentration.

Std. Dev.: standard deviation.

Table 6.1.5/2: Mean specific growth rate in P. subcapitata during the final test

	Replicate	Nominal concentration and corresponding measured concentration (geometric mean)  (mg test item.L^-1)
		Control (0)	0.5 (0.81)	1.0 (1.01)	2.0 (2.08)	4.0 (4.44)	8.0 (8.75)
t=0h - t=24h	1	1.856	1.723	1.856	1.723	1.163	0.875
	2	2.079	2.079	2.079	2.175	0.470	-0.223
	3	2.485	1.974	1.974	1.856	1.723	-1.609
	4	2.485
	5	1.723
	6	2.175
	Mean	2.134	1.925	1.970	1.918	1.119	-0.319
	Std. Dev.	0.3153	0.1832	0.1116	0.2322	0.6276	1.2452
	% Inh.	-	9.8	7.7	10.1	47.6	115.0
t=0h - t=48h	1	1.844	1.707	1.745	1.733	1.275	0.582
	2	1.781	1.834	1.733	1.555	1.040	0.235
	3	1.901	1.781	1.781	1.456	1.208	-0.112
	4	1.864
	5	1.733
	6	1.792
	Mean	1.819	1.774	1.753	1.581	1.174	0.235
	Std. Dev.	0.0617	0.0638	0.0247	0.1403	0.1211	0.3466
	% Inh.	-	2.5	3.6	13.1	35.5	87.1
t=0h - t=72h	1	1.454	1.374	1.440	1.444	1.024	-0.074
	2	1.548	1.365	1.433	1.322	0.870	-0.074
	3	1.587	1.390	1.342	1.322	0.940	-0.536
	4	1.602
	5	1.521
	6	1.566
	Mean	1.546	1.376	1.405	1.363	0.945	-0.228
	Std. Dev.	0.0535	0.0130	0.0548	0.0701	0.0772	0.2668
	% Inh.	-	11.0	9.1	11.9	38.9	114.8

Values were extracted from the computer program ToxRat

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

Table 6.1.5/3: Concentrations of the test substance (mg/L) in test water - final test

Nominal concentration (mg.L^-1)	Measured concentration (mg.L^-1)		Geometric mean measured concentrations
	Start(t=0h)	End(t=72h)	mg.L^-1	% nominal
Control	Abs.	Abs.	N.A.	N.A.
0.5	0.79	0.83	0.81	162
1.0	0.96	1.06	1.01	101
2.0	2.18	1.99	2.08	104
4.0	4.30	4.58	4.44	111
8.0	8.79	8.72	8.75	109

N.A.: not applicable

% = Percent of expected nominal concentration in test item.

Abs.= Absence: concentrations below the LOQ (0.45 mg.L^-1) and the LOD (0.13 mg.L^-1).

Validity criteria fulfilled:

yes

Remarks:

See Overall remarks

Conclusions:

The toxic effect of test item to the unicellular algal species Pseudokirchneriella subcapitata was investigated in a closed static test. Under the experimental conditions and based on the geometric means of measured exposure concentrations, the 72h-ErC50 and 72h-ErC10 values for the parameter growth rate were determined to be 4.845 mg/L and 2.256 mg/L, respectively. Since the 72h-NOEC value for growth rate could not be determined, the endpoint values at t=48h were also determined, for information only, in order to have a complete set NOEC/EC10/EC50 (validity criteria of the study at t=48h were also respected). The 48d-ErC50 and 48h-ErC10 values for the parameter growth rate were determined to be 5.369 mg/L and 2.058 mg/L, respectively. The 48h-NOEC value for growth rate was 1.010 mg/L.

Executive summary:

A study was performed to assess the test substance for its ability to generate toxic effects in the unicellular algal species Pseudokirchneriella subcapitata, according to OECD Guideline 201 and EU Method C3 with GLP compliance. Following a preliminary range-finding test, algal cells were exposed to an aqueous solution of

the test substance over 72 hours at the required nominal test concentrations 0.5, 1.0, 2.0, 4.0 and 8.0 mg/L, under closed and static conditions. 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 the end of the test in order to determine if concentrations of the test item were maintained. These chemical analysis revealed that test item levels were stable, with losses of test item < 20% compared to initial values. Furthermore, test item concentrations were satisfactorily maintained within ± 20% of the nominal concentrations throughout the test, except for the geometric mean of the measured concentrations at 0.5 mg/L which was higher than the corresponding nominal concentration over the study period (with a geometric mean of 162%). Due to this single concentration difference, the evaluation of the effects on P. subcapitata was based on the geometric means of the measured concentrations.

Under the experimental conditions and based on the geometric means of measured exposure concentrations, the 72h-ErC50 and 72h-ErC10 values for the parameter growth rate were determined to be 4.845 mg/L and 2.256 mg/L, respectively. Since the 72h-NOEC value for growth rate could not be determined, the endpoint values at t=48h were also determined, for information only, in order to have a complete set NOEC/EC10/EC50. Indeed, according to OECD 201 section 11, it is possible to shorten the test period to 48 hours whenever unlimited, exponential growth during the test has been obtained. This corresponds to at least a 16-fold growth in controls. Since the cell density in the control cultures increased by a factor of 38 within 48 hours exposure period, the results can also be provided after 48h according to OECD 201. The 48d-ErC50 and 48h-ErC10 values for the parameter growth rate were determined to be 5.369 mg/L and 2.058 mg/L, respectively. The 48h-NOEC value for growth rate was 1.010 mg/L.

Description of key information

OECD Guideline 201, EU Method C3, GLP, key study, validity 1:

72h-ErC50 (P. subcapitata) = 4.845 mg/L (95% CL: 4.329 - 5.432 mg/L), based on geometric mean measured concentrations.

72h-ErC10 (P. subcapitata) = 2.256 mg/L (95% CL: 1.523 - 2.781 mg/L), based on geometric mean measured concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:

4.85 mg/L

EC10 or NOEC for freshwater algae:

2.26 mg/L

Additional information

One experimental study is available to assess the registered substance for its ability to generate toxic effects in the unicellular algal species Pseudokirchneriella subcapitata, according to OECD Guideline 201 and EU Method C3 with GLP compliance. Following a preliminary range-finding test, algal cells were exposed to an aqueous solution of

the test substance over 72 hours at the required nominal test concentrations 0.5, 1.0, 2.0, 4.0 and 8.0 mg/L, under closed and static conditions. 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 the end of the test in order to determine if concentrations of the test item were maintained. These chemical analysis revealed that test item levels were stable, with losses of test item < 20% compared to initial values. Furthermore, test item concentrations were satisfactorily maintained within ± 20% of the nominal concentrations throughout the test, except for the geometric mean of the measured concentrations at 0.5 mg/L which was higher than the corresponding nominal concentration over the study period (with a geometric mean of 162%). Due to this single concentration difference, the evaluation of the effects on P. subcapitata was based on the geometric means of the measured concentrations.

Under the experimental conditions and based on the geometric means of measured exposure concentrations, the 72h-ErC50 and 72h-ErC10 values for the parameter growth rate were determined to be 4.845 mg/L and 2.256 mg/L, respectively. Since the 72h-NOEC value for growth rate could not be determined, the endpoint values at t=48h were also determined, for information only, in order to have a complete set NOEC/EC10/EC50. Indeed, according to OECD 201 section 11, it is possible to shorten the test period to 48 hours whenever unlimited, exponential growth during the test has been obtained. This corresponds to at least a 16-fold growth in controls. Since the cell density in the control cultures increased by a factor of 38 within 48 hours exposure period, the results can also be provided after 48h according to OECD 201. The 48d-ErC50 and 48h-ErC10 values for the parameter growth rate were determined to be 5.369 mg/L and 2.058 mg/L, respectively. The 48h-NOEC value for growth rate was 1.010 mg/L.