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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:

07-10-2019 to 03-12-2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, 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:

Inspected on 29-30-31 May 2018 / Signature date: 15 November 2018

Analytical monitoring:

yes

Details on sampling:

Samples taken from the control and all test concentrations(from a replicate of each test concentration with algae dedicated exclusively to chemical analyses) 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.

Vehicle:

no

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata, CCAP 278/4

Origin: 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.

Reason for selection : This system is a unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.

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.
Then, the mixture was made up to 1000 mL with sterilised water. The pH of this solution was approximately in the range of 8.1 ± 0.2.

Light regime :Continuous illumination

Light intensity :4 440-8 880 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 1.104 cells.mL-1. 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 23.2 and 23.7°C throughout the test.

pH:

See Table 6.1.5/1 in "Any other information on results incl. tables"
Although the pH level in the control varied by more than 1.5 units at the end of the test (1.51 units of difference) this was not considered to have affected the integrity of the study. The cause of the pH increases in the control 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.

Nominal and measured concentrations:

- Nominal Loading Rates: 0.40, 0.67, 1.12, 1.86 and 3.11 mg/L.
- Specific analyses of samples taken from all test concentrations revealed unavoidable test item losses. These decreases were inversely proportional to the tested concentrations, suggesting potential adsorption/metabolism effects by algae at the (non-toxic) lowest concentrations. Since the deviation of the exposure concentrations of the test substance was greater than ± 20% of the initial concentrations, the results were based on the geometric means of measured exposure concentrations.
- Measured concentrations: See tables 6.1.5/2 in "Any other information on results incl. tables".

Details on test conditions:

TEST SYSTEM
- Test type : A static test was performed.
- Test vessel: 100 mL, all-glass closed flasks with ground glass stopper, completely filled with test solution with minimum headspace.
Each test vessel was uniquely identified with study code, replicate number, date of experimentation and treatment group
- Cell concentration : An initial cell density of 5000 cells.mL-1 using the exponentially growing pre-culture.
- Replicates : 6 controls and 3 replicates of each test concentration for counting. Moreover, additional replicates of each treatment with algae were prepared for chemical analyses (destructive samples) in order to determine maintenance of actual concentrations.


GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
-Test water : Original medium of 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 were added to the sterilised water during test water reconstitution (instead of 1 mL) to obtain a final concentration of 350 mg.L-1, according to the study plan.


OTHER TEST CONDITIONS
-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.

- Light regime/intensity :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) :
- Cell densities 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.
- 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 conditions were recorded with suitable instruments and documented in the raw data.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.67

RANGE FINDING STUDY :
A range-finding test was conducted between July 1 and July 5, 2019 to determine the range
of concentrations for the definitive test. The raw data of this test are archived under the project number of the present study.
The mixing vessel was a cylindrical glass bottle sealed with 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. 0.56 g) 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 24 hours of gentle stirring in the dark and at room temperature, the contents of the vessel were allowed to stand undisturbed for at least 1 hour before use. The first 100 mL of solution was discarded via the drain port. Samples were taken from the saturated 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 (11.43 mg.L-1), and a fixed amount of inoculum in order to obtain the required test concentrations in the test vessels and a cell density of 5.103 cells.mL-1 per vessel. The test vessels were completely filled (with minimum headspace) and were closed with ground glass stoppers. At the start of the test, test solutions in all test vessels were observed to be clear and colourless at all test concentrations. The test was carried out without adjustment of the pH.
In this range-finding test, algal cells were exposed to a series of nominal test concentrations of 0.32, 1.00, 3.20 mg test item.L-1 and to a concentration close to the solubility limit of the test item in test water (11.43 mg.L-1, slightly diluted by the algae addition : 10.29 mg.L-1), 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.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.929 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I.: 1.818-2.055 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.895 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I.: 0.775-0.998 mg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.335 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate

Remarks on result:

other: 95% C.I.: 2.120-2.641 mg/L

Details on results:

- Analytical results: See tables 6.1.5/3 and 4 in "Any other information on results incl. tables" (Table 3. Yield of algal cells during the final test ; Table 4. Mean specific growth rate in P. subcapitata during the final test.

Results with reference substance (positive control):

On August 26, 2019 (KA19-002; most recent test), the 72h-EC50 was 0.719 mg.L-1 for the parameter growth rate. Hence, the sensitivity of this batch of Pseudokirchneriella subcapitata was consistent with the level proposed by the ISO 8692 (6) (expected 72h-ErC50: 0.65 mg.L-1 to 1.73 mg.L-1).

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.

Table 6.1.5/1. pH-values during the final test.

 

	Nominal concentration and corresponding measured concentration (geometric mean) after 48 h and after 72 h (mg test item.L^-1)
	Control (0) (0)	0.40 (0.37) (0.22)	0.67 (0.63) (0.53)	1.12 (1.04) (0.89)	1.86 (1.68) (1.49)	3.11 (2.70) (2.66)
Start t=0 h	8.31	8.24	8.23	8.22	8.26	8.28
End t=72 h	9.82	9.92	9.80	9.63	9.00	8.51

 

 

Table 6.1.5/2 .Concentrations of the test item (mg.L^-1) in test water - Final test.

Nominal concentration (mg.L^-1)	Measured concentration (mg.L^-1)				Relative loss to initial value (% initial)		Geometric mean measured concentrations
	Start (t=0 h)	t=24 h	t=48 h	t=72 h
							mg.L^-1		% nominal
					(t=0 h - t=48 h)	(t=0 h - t=72 h)	(t=0 h - t=48 h)	(t=0 h - t=72 h)	(t=0 h - t=48 h)	(t=0 h - t=72 h)
Control	Abs.	Abs.	Abs.	Abs.	N.A.	N.A.	N.A.	N.A.	N.A.	N.A.
0.40	0.45	0.40	0.28	Pres.*	38	N.A.	0.37	0.22	93	55
0.67	0.71	0.67	0.52	0.31	27	56	0.63	0.53	94	79
1.12	1.21	1.09	0.85	0.57	30	53	1.04	0.89	93	79
1.86	1.99	1.85	1.28	1.04	36	48	1.68	1.49	90	80
3.11	3.41	3.11	1.85	2.56	46	25	2.70	2.66	87	86

_{N.A.: not applicable}

_{% = Percent of expected nominal concentration in test item.}

_{*Pres= Presence: concentrations below the LOQ (0.10 mg.L-1) but above the LOD (0.03 mg.L-1). Where measured concentrations were below the LOQ (0.10 mg.L-1) but above the LOD (0.03 mg.L-1), such concentrations were considered to be the half of the quantification limit, according to the study plan (à LOQ/2 = 0.05 mg.L-1).}

_{Abs= Absence: concentrations below the LOQ (0.10 mg.L-1) and the LOD (0.03 mg.L-1).}

 

Table 6.1.5/3. Yield of algal cells during the final test.

	Replicate	Nominal concentration and corresponding measured concentration (geometric mean) after 48 h and after 72 h (mg test item.L^-1)
		Control (0) (0)	0.40 (0.37) (0.22)	0.67 (0.63) (0.53)	1.12 (1.04) (0.89)	1.86 (1.68) (1.49)	3.11 (2.70) (2.66)
t=24 h	1	2.7	0.7	1.5	1.9	1.9	0.3
	2	4.7	3.1	1.9	2.7	2.3	0.3
	3	4.3	3.5	2.3	1.5	2.3	0.7
	4	5.5
	5	5.9
	6	2.7
	Mean	4.3	2.4	1.9	2.0	2.2	0.4
	Std. Dev.	1.36	1.51	0.40	0.61	0.23	0.23
	% Red.	-	43.4	55.8	52.7	49.6	89.9
t=48 h	1	19.9	20.7	13.9	14.7	3.9	2.3
	2	19.5	21.5	18.3	10.3	4.7	3.1
	3	17.1	18.7	17.5	11.5	5.5	1.1
	4	21.5
	5	17.5
	6	20.7
	Mean	19.4	20.3	16.6	12.2	4.7	2.2
	Std. Dev.	1.75	1.44	2.34	2.27	0.80	1.01
	% Red.	-	-4.8	14.5	37.2	75.7	88.8
t=72 h	1	83.5	87.5	61.9	53.9	16.3	1.5
	2	95.5	81.1	63.5	58.3	15.9	2.7
	3	75.5	74.3	58.7	44.3	18.7	1.1
	4	99.9
	5	90.7
	6	85.5
	Mean	88.4	81.0	61.4	52.2	17.0	1.8
	Std. Dev.	8.80	6.60	2.44	7.16	1.51	0.83
	% Red.	-	8.4	30.6	41.0	80.8	98.0

_{Values were extracted from the computer program ToxRat (8).}

_{% Red.: %Reduction in yield relative to the control determined by ToxRat (8).}

 

Table 6.1.5/4. Mean specific growth rate in P. subcapitata during the final test.

	Replicate	Nominal concentration and corresponding measured concentration (geometric mean) after 48 h and after 72 h (mg test item.L^-1)
		Control (0) (0)	0.40 (0.37) (0.22)	0.67 (0.63) (0.53)	1.12 (1.04) (0.89)	1.86 (1.68) (1.49)	3.11 (2.70) (2.66)
t=0 h - t=24 h	1	1.856	0.875	1.386	1.569	1.569	0.470
	2	2.342	1.974	1.569	1.856	1.723	0.470
	3	2.262	2.079	1.723	1.386	1.723	0.875
	4	2.485
	5	2.549
	6	1.856
	Mean	2.225	1.643	1.559	1.604	1.671	0.605
	Std. Dev.	0.3032	0.6668	0.1684	0.2370	0.0890	0.2341
	% Inh.	-
t=0 h - t=48 h	1	1.854	1.874	1.680	1.707	1.087	0.861
	2	1.844	1.892	1.814	1.536	1.171	0.987
	3	1.781	1.824	1.792	1.589	1.242	0.582
	4	1.892
	5	1.792
	6	1.874
	Mean	1.839	1.863	1.762	1.611	1.167	0.810
	Std. Dev.	0.0446	0.0352	0.0715	0.0875	0.0776	0.2076
	% Inh.	-
t=0 h - t=72 h	1	1.708	1.723	1.609	1.563	1.172	0.462
	2	1.752	1.698	1.617	1.589	1.163	0.619
	3	1.675	1.669	1.591	1.498	1.216	0.388
	4	1.767
	5	1.735
	6	1.716
	Mean	1.726	1.697	1.606	1.550	1.184	0.490
	Std. Dev.	0.0334	0.0271	0.0133	0.0467	0.0283	0.1179
	% Inh.	-

Values were extracted from the computer program ToxRat (8).

% Inh.: %Inhibition of growth rate relative to the control determined by ToxRat (8).

 

              

Validity criteria fulfilled:

yes

Conclusions:

Under the experimental conditions and based on the geometric means of measured exposure concentrations, the 72-hour EC50 for the parameters growth rate awere determined to be 1.929 mg.L-1y. The 72-hour EC10 for growth rate was 0.895 mg.L-1.

Executive summary:

A study was performed in a closed static test to assess the test item ST 08 C 19 for its ability to generate toxic effects in the unicellular algal species Pseudokirchneriella subcapitata. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", referenced as Method C.3 of Commission Regulation No. 440/2008 and with the “Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (OECD No. 23).

Following a preliminary range-finding test, algal cells were exposed to an aqueous solution of the test item over 72 hours at the required nominal test concentrations 0.40, 0.67, 1.12, 1.86 and 3.11 mg.L^-1. 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 analyses of samples taken from all test concentrations revealed unavoidable test item losses. These decreases were inversely proportional to the tested concentrations, suggesting potential adsorption/metabolism effects by algae at the (non-toxic) lowest concentrations. Since the deviation of the exposure concentrations of the test substance was greater than ± 20% of the initial concentrations, the results were based on the geometric means of measured exposure concentrations.

 

Under the experimental conditions and based on the geometric means of measured exposure concentrations, the 72-hour EC50 for the parameters growth rate were determined to be 1.929 mg.L-1. The 72-hour EC10 for growth rate was 0.895 mg.L-1.

Description of key information

OECD Guideline 201, EU Method C.3, GLP, Key study, validity 1:

72h-EC50 (Pseudokirchneriella subcapitata) = 1.929 mg/L (95% CL: 1.818 - 2.055 mg/L)

72h-EC10 (Pseudokirchneriella subcapitata) = 0.895 mg/L (95% CL: 0.775 - 0.998 mg/L)

Key value for chemical safety assessment

EC50 for freshwater algae:

1.929 mg/L

EC10 or NOEC for freshwater algae:

0.895 mg/L

Additional information

A study was performed in a closed static test to assess the test item ST 08 C 19 for its ability to generate toxic effects in the unicellular algal species Pseudokirchneriella subcapitata. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", referenced as Method C.3 of Commission Regulation No. 440/2008 and with the “Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (OECD No. 23).

Following a preliminary range-finding test, algal cells were exposed to an aqueous solution of the test item over 72 hours at the required nominal test concentrations 0.40, 0.67, 1.12, 1.86 and 3.11 mg.L^-1. 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 analyses of samples taken from all test concentrations revealed unavoidable test item losses. These decreases were inversely proportional to the tested concentrations, suggesting potential adsorption/metabolism effects by algae at the (non-toxic) lowest concentrations. Since the deviation of the exposure concentrations of the test substance was greater than ± 20% of the initial concentrations, the results were based on the geometric means of measured exposure concentrations.

 

Under the experimental conditions and based on the geometric means of measured exposure concentrations, the 72-hour EC50 for the parameters growth rate were determined to be 1.929 mg.L-1. The 72-hour EC10 for growth rate was 0.895 mg.L-1.