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Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23rd July 2021 - 4th October 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study carried out according to OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 761/2009 with no deviation from guidance. In accordance with GLP.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

761/2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection 11th August - 30th September, date on certificate 14th January 2022

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were analyzed on the day of sampling. Duplicate samples were taken on each occasion and stored frozen for further analysis if necessary.
An aliquot of each sample was analysed directly or diluted with media.

Vehicle:

no

Details on test solutions:

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g., ultrasonication and high shear mixing. The test item was however found to be soluble with the aid of prolonged stirring. A nominal amount of the test item was dispersed in culture medium with the aid of magnetic stirring for 24 hours to give a stock solution (100 mg/L) at the beginning of the experiment.

The culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl. The culture medium used for the range-finding and definitive tests was the same as that used to maintain the stock culture.
A nominal amount of test item (222 mg) was dispersed in 2 L of culture medium with the aid of magnetic stirring for 24 hours to give a stock solution at a concentration of 100 mg/L.
Following stirring the preparation was allowed to stand for 1-Hour prior to removal of the aqueous phase by mid-depth siphon. A series of dilutions was made from this stock solution to give further stock solutions of 32, 10, 3.2, 1.0 and 0.32 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with 2.2 mL of algal suspension to give an initial nominal cell density of 5.00 x 10^3 cells/mL. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.

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): The Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.

ACCLIMATION
Approximately 3 to 4 days before the start of the test, inoculum cultures of algae were set up at an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^5 to 10^6 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

No data

Test temperature:

24 ± 1 °C

pH:

Control: 7.5 at 0 h, 8.1 at 72 h. 0.32, 1.0 3.2, 10 mg ai/L:7.1-7.5 at 0 h, 8.2-8.4 at 72 h. 32 mg ai/L: 5.3 at 0 h, 6.2 at 72 h; 100 mg ai/L: 3.5 at 0 h, 3.6 at 72 h.
Given that this was considered to be due to an intrinsic property of the test item, no adjustments to pH were made prior to inoculation with algae at the start of the test.

Dissolved oxygen:

No data

Salinity:

Not Applicable

Conductivity:

No data

Nominal and measured concentrations:

Measured concentrations fell within an acceptable range (80-120%) of the nominal concentrations for most of the measured samples apart from the 72 h 0.32 and 32 mg/L (121% and 127%, respectively) samples from the definitive test. For full data see Tables 6.1 and 6.2 in the Any other information on Materials and Methods inc. tables section

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL, glass conical flasks with 100 mL solution in each.
- Type (delete if not applicable): Closed
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable.
- Renewal rate of test solution (frequency/flow rate): Not applicable.
- Initial cells density: 5000 cells/mL.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable.
- Test environment: The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron incubator) at 24 °C ± 1 °C; The flasks were constantly shaken at approximately 150 rpm for 72 hours.

GROWTH MEDIUM
- Standard medium used: yes. The contents can be seen in the Any other information on materials and methods incl. tables section.

TEST MEDIUM / WATER PARAMETERS
The same culture medium that was used for growth medium was used for the experiments.

OTHER TEST CONDITIONS
- Sterile test conditions: not reported.
- Adjustment of pH: The pH of the test solutions was not adjusted.
- Photoperiod: Continuous illumination.
- Light intensity: approx 7000 lux (provided by warm white lighting (380 to 730 nm)).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Cell densities (samples were taken at 23, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not applicable (limit test).
- Range finding study: Yes
- Test concentrations in the range-finding tests: 0, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: The results showed growth was observed to be reduced at 1.0, 10 and 100 mg/L. Based on this information test concentrations of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

A positive control using potassium dichromate as the reference item at concentrations of 0.125, 0.25, 0.50, 1.0 and 2.0 mg/L was conducted.

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

25 mg/L

95% CI:

4.1 - 28

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

17 mg/L

95% CI:

9.6 - 782

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 6.5. Daily specific growth rates for the control cultures are given in Table 6.6. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 6.7.
The mean cell densities versus time for the definitive test are presented in Figure 1. Percentage inhibition values are plotted against test concentration in Figure 6.4 and Figure 6.5.
From the data given in Table 6.4 and Table 6.5, it is clear that the growth rate (r) and yield (y) of Raphidocelis subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.

Accordingly the following results were determined from the data:
ErC10 (0 to 72 hour): 15 mg/L; 95% confidence limits 0.060 to 22 mg/L
ErC20 (0 to 72 hour): 18 mg/L; 95% confidence limits 0.26 to 24 mg/L
ErC50 (0 to 72 hour): 25 mg/L; 95% confidence limits 4.1 to 28 mg/L

Where ErCx is the test concentration that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and all test concentrations using Williams Multiple Sequential t-test Procedure. There were no statistically significant differences between the control, 0.32, 1.0, 3.2 and 10 mg/L test concentrations (P≥0.05); however, all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on growth rate was 10 mg ai/L. Correspondingly the LOEC based on growth rate was 32 mg/L.


EyC10 (0 to 72 hour): 11 mg ai/L; 95% confidence limits 0.50 to 22 mg ai/L
EyC20 (0 to 72 hour): 13 mg ai/L; 95% confidence limits 2.5 to 40 mg ai/L
EyC50 (0 to 72 hour): 17 mg ai/L; 95% confidence limits 9.6 to 782 mg ai/L
Where EyCx is the test concentration that reduced yield by x%.

There were no statistically significant differences between the control, 0.32, 1.0, 3.2 and 10 mg ai/L test concentrations (P≥0.05); however, all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on yield was 10 mg ai/L. Correspondingly the LOEC based on yield was 32 mg ai/L.

Results with reference substance (positive control):

Exposure of Raphidocelis subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 to 72 hour) : 1.3 mg/L; 95% confidence limits 1.2 to 1.5 mg/L
EyC50 (0 to 72 hour) : 0.44 mg/L; 95% confidence limits 0.37 to 0.52 mg/L

No Observed Effect Concentration based on growth rate:0.125 mg/L
No Observed Effect Concentration based on yield: 0.125 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.25 mg/L
Lowest Observed Effect Concentration based on yield: 0.25 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item as tested in previous in-house experiments (data from the previous five studies shows a mean ErC50 value of 1.4 mg/L
(standard deviation = 0.16) and a mean EyC50 value of 0.64 mg/L (standard deviation = 0.12)).

Reported statistics and error estimates:

All ECx values and associated confidence limits and the slope of the response curve and its standard error for growth rate and yield were calculated by Probit analysis using Linear Maximum-Likelihood regression. All ECx values were determined using the ToxRat Professional computer software package.

Replicate R1 of the 0.32 mg ai/L test group was accidentally broken and the contents lost following sampling for cell density determinations at 48 hours. As such the data for this replicate was excluded from all statistical analyses.

Table 6.3. Effects of the test item on the growth and yield of Raphidocelis subcapitata

Response Variable	EC50  (mg ai/L)	95% Confidence Limits (mg ai/L)	No Observed Effect Concentration (NOEC) (mg ai/L)	Lowest Observed Effect Concentration (LOEC) (mg ai/L)
Growth	25	4.1 - 28	10	32
Yield	17	9.6 - 782	10	32

 

 

Table 6.4. Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

 

Nominal Concentrations       (mg ai/L)		Cell Densities* (cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth rate	Yield
Control	R1	5.22E+03	6.81E+05	-	-
	R2	5.66E+03	8.43E+05
	Mean	5.44E+03	7.62E+05
1.0	R1	6.86E+03	6.26E+05	9	11
	R2	7.77E+03	7.36E+05
	Mean	7.32E+03	6.81E+05
10	R1	8.36E+03	4.02E+05	13	34
	R2	5.43E+03	6.12E+05
	Mean	6.89E+03	5.07E+05
100	R1	7.66E+03	2.39E+04	86	99
	R2	6.86E+03	5.87E+03
	Mean	7.26E+03	1.49E+04

*  Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks

R -  = Replicate
-      = Not applicable

 

 

 

Table 6.5. Cell Densities and pH Values in the Definitive Test

Nominal Concentration (mg ai/L)		pH	Cell Densities* (cells per mL)			pH
		0 Hours	23 Hours	48 Hours	72 Hours	72 Hours
Control	R1	7.5	2.42E+04	1.05E+05	5.14E+05	8.1
	R2		2.25E+04	1.08E+05	5.68E+05
	R3		2.50E+04	1.28E+05	7.39E+05
	R4		2.29E+04	1.27E+05	5.84E+05
	R5		2.88E+04	1.48E+05	5.82E+05
	R6		2.88E+04	1.37E+05	5.90E+05
	Mean		2.54E+04	1.25E+05	5.96E+05
0.32	R1	7.5	2.60E+04	9.40E+04	**	8.3
	R2		2.82E+04	1.51E+05	5.41E+05
	R3		2.81E+04	1.60E+05	6.80E+05
	Mean		2.74E+04	1.35E+05	6.06E+05
1.0	R1	7.5	2.73E+04	1.08E+05	6.24E+05	8.4
	R2		2.34E+04	1.23E+05	6.48E+05
	R3		2.72E+04	1.05E+05	6.52E+05
	Mean		2.59E+04	1.12E+05	6.42E+05
3.2	R1	7.4	2.19E+04	9.00E+04	4.04E+05	8.2
	R2		2.41E+04	1.16E+05	6.05E+05
	R3		2.50E+04	1.14E+05	5.65E+05
	Mean		2.36E+04	1.07E+05	5.24E+05
10	R1	7.1	2.31E+04	1.29E+05	5.93E+05	8.3
	R2		1.94E+04	1.05E+05	5.79E+05
	R3		2.02E+04	1.27E+05	5.36E+05
	Mean		2.09E+04	1.20E+05	5.69E+05
32	R1	5.3	8.51E+03	1.07E+04	2.69E+04	6.2
	R2		1.65E+04	8.18E+03	2.24E+04
	R3		5.57E+03	4.87E+03	8.80E+03
	Mean		1.02E+04	7.91E+03	1.94E+04
100	R1	3.5	3.23E+03	2.08E+03	2.29E+03	3.6
	R2		3.81E+03	4.61E+03	4.55E+03
	R3		5.28E+03	2.93E+03	3.78E+03
	Mean		4.11E+03	3.21E+03	3.54E+03

*  Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

**  Flask broken and sample lost following the removal of an aliquot for cell density determinations at 48 hours. As such results for this replicate have not been determined, mean results for this test group calculated based on replicates 2 and 3 only.

R = Replicate

 

 

 

 

Table 6.6. Daily Specific Growth Rates for the Control Cultures in the Definitive Test

Treatment		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 to 1	Day 1 to 2	Day 2 to 3
Control	R1	0.069	0.059	0.066
	R2	0.065	0.063	0.069
	R3	0.070	0.065	0.073
	R4	0.066	0.068	0.064
	R5	0.076	0.065	0.057
	R6	0.076	0.063	0.061
	Mean	0.070	0.064	0.065

 

Table 6.7. Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration (mg ai/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 Hours	% Inhibition	0 to 72 Hours	% Inhibition*
Control	R1	0.064	-	5.09E+05	-
	R2	0.066		5.63E+05
	R3	0.069		7.34E+05
	R4	0.066		5.79E+05
	R5	0.066		5.77E+05
	R6	0.066		5.85E+05
	Mean	0.066		5.91E+05
	SD	0.002		7.51E+04
0.32	R1	**		**
	R2	0.065	2	5.36E+05
	R3	0.068	[3]	6.75E+05
	Mean	0.067	[1]	6.06E+05	[3]
	SD	0.001		9.83E+04
1.0	R1	0.067	[2]	6.19E+05
	R2	0.068	[3]	6.43E+05
	R3	0.068	[3]	6.47E+05
	Mean	0.068	[3]	6.37E+05	[8]
	SD	0.001		1.52E+04
3.2	R1	0.061	8	3.99E+05
	R2	0.067	[2]	6.00E+05
	R3	0.066	0	5.60E+05
	Mean	0.065	2	5.19E+05	12
	SD	0.003		1.06E+05
10	R1	0.066	0	5.88E+05
	R2	0.066	0	5.74E+05
	R3	0.065	2	5.31E+05
	Mean	0.066	1	5.64E+05	5
	SD	0.001		3.00E+04
32	R1	0.023	65	2.19E+04
	R2	0.021	68	1.74E+04
	R3	0.008	88	3.80E+03
	Mean	0.017	74	1.44E+04	98
	SD	0.008		9.41E+03
100	R1	-0.011	117	-2.71E+03
	R2	-0.001	102	-4.53E+02
	R3	-0.004	106	-1.22E+03
	Mean	-0.005	108	-1.46E+03	100
	SD	0.005		1.15E+03

 

* In accordance with the OECD test calculated guideline only the mean value for yield for each test concentration is calculated

R = Replicate
- = Not applicable
SD = Standard Deviation
** Flask broken and sample lost following the removal of an aliquot for cell density determinations at 48 hours. As such results for this replicate have not been determined, mean and standard deviation results for this test group calculated based on replicates 2 and 3 only.

 [ ] = Increase in growth compared to controls

Validity criteria fulfilled:

yes

Remarks:

The following data show that the cell concentration of the control cultures increased by a factor of 119 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Conclusions:

The effect of the test item on the growth of Raphidocelis subcapitata has been investigated over a 72 hour period and based on the nominal test concentrations gave the following results: EC50: 25 mg/L (growth), 17 mg/L (yield); NOEC: 10 mg/L (growth and yield); LOEC: 32 mg/L (growth and yield).

Executive summary:

A study was performed to assess the test substance for its ability to generate toxic effects in the unicellular algal species Raphidocelis subcapitata, according to OECD Test Guideline 201 and EU Method C.3, with GLP compliance. 

 

A limit test was performed following the results of a range-finding test. During the range-finding test, algal cells were exposed to the test substance at nominal concentrations of 1.0, 100 mg/L and to a control. Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L. Samples were taken at 23, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. The nominal inoculated cell concentration (5.00 x 103 cells/mL) was taken as the starting cell density.

 

A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. Majority of the measured concentrations fell within the 80-120% range of nominal concentrations, therefore nominal concentrations were used for calculating the results.

 

The test item affected both the growth rate and the yield of the algae. For the growth rate, the effects can be summarised thus:

 

ErC10 (0 to 72 hour): 15 mg/L; 95% confidence limits 0.060 to 22 mg/L

ErC20 (0 to 72 hour): 18 mg/L; 95% confidence limits 0.26 to 24 mg/L

ErC50 (0 to 72 hour): 25 mg/L; 95% confidence limits 4.1 to 28 mg/L

(ErCx is the test concentration that reduced growth rate by x%.)

 

And the effects on the yield were the following:

 

EyC10 (0 to 72 hour): 11 mg/L; 95% confidence limits 0.50 to 22 mg ai/L

EyC20 (0 to 72 hour): 13 mg/L; 95% confidence limits 2.5 to 40 mg ai/L

 EyC50 (0 to 72 hour): 17 mg/L; 95% confidence limits 9.6 to 782 mg ai/L

 

In conclusion, the test substance was found to have an adverse effect on the growth and yield of the algae Raphidocelis subcapitata (LOEC = 32 mg/L; NOEC = 10 mg/L).

Description of key information

A study was performed to assess the test substance for its ability to generate toxic effects in the unicellular algal species Raphidocelis subcapitata, according to OECD Test Guideline 201 and EU Method C.3, with GLP compliance. 

A limit test was performed following the results of a range-finding test. During the range-finding test, algal cells were exposed to the test substance at nominal concentrations of 1.0, 100 mg/L and to a control. Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L. Samples were taken at 23, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. The nominal inoculated cell concentration (5.00 x 103 cells/mL) was taken as the starting cell density.

A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. Majority of the measured concentrations fell within the 80-120% range of nominal concentrations, therefore nominal concentrations were used for calculating the results.

The test item affected both the growth rate and the yield of the algae. For the growth rate, the effects can be summarised thus:

EC10 (0 to 72 hour): 15 mg/L; 95% confidence limits 0.060 to 22 mg/L

EC20 (0 to 72 hour): 18 mg/L; 95% confidence limits 0.26 to 24 mg/L

EC50 (0 to 72 hour): 25 mg/L; 95% confidence limits 4.1 to 28 mg/L

And the effects on the yield were the following:

EC10 (0 to 72 hour): 11 mg/L; 95% confidence limits 0.50 to 22 mg/L

EC20 (0 to 72 hour): 13 mg/L; 95% confidence limits 2.5 to 40 mg/L

EC50 (0 to 72 hour): 17 mg/L; 95% confidence limits 9.6 to 782 mg/L

In conclusion, the test substance was found to have an adverse effect on the growth and yield of the algae Raphidocelis subcapitata (LOEC = 32 mg/L; NOEC = 10 mg/L).

Key value for chemical safety assessment

EC50 for freshwater algae:

17 mg/L

EC10 or NOEC for freshwater algae:

10 mg/L