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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:

19 February 2018 to 24 February 2018

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)

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The content of DOC in the test vessels was measured at the start and at the end of the test.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- The water-accommodated fraction (WAF) was prepared for the test. This was done by mixing 10.0 mg/L with the corresponding amount of algal medium (demineralised water enriched with minerals but without algae) and shaking for 24 hours. The resulting solution was filtrated through 0.45 μm PTFE filters.
- The lower treatments were prepared by dilution of this WAF with nutrient medium. The resulting solutions were used for preparation of the treatments.
- This procedure is in agreement with the OECD guidance document no. 23 for the testing of mixtures which are toxic at very low loading rates. In a non-GLP pre-test, at the loading rate of 1 mg/L, inhibition of algal growth was observed.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: 86.81
- Source: The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sci-encebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

ACCLIMATION
- Four days before start of the experiment, an aliquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 96 hours (5 000 lux, 21.6 – 22.8 °C). The resulting culture grew exponentially.
- Before usage, the pre-culture was checked for the absence of cell aggregates and the cell number of culture was determined.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21.5 – 23.1 °C

pH:

7.6 - 8.2

Nominal and measured concentrations:

Nominal: 0.032, 0.1, 0.32, 1, 3.2 and 10 mg/L
Calculated concentrations: 0.011, 0.035, 0.111, 0.346, 1.108 and 3.462 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass flasks, total volume 65 mL.
- Type: Open, covered with perforated plastic foil acting as a stopper.
- Flasks were shaken on an orbital shaker to keep the algae in suspension.
- Material, size, headspace, fill volume: 45 ± 1 mL
- Initial cells density: For each treatment, 200 mL of the respective test material solution was mixed with the necessary amount of algal pre-culture (0.360 mL) to achieve a cell concentration of 2.4*10^3 cells/mL. For the blank control, 350 mL nutrient medium was used instead of test material solution and mixed with the necessary amount of algal pre-culture (0.630 mL).
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Algal medium: Stock solution I: 10.0 mL; Stock solution II: 1.0 mL; Stock solution III: 1.0 mL; Stock solution IV: 1.0 mL and H2O deionised ad 1 000 mL.
Stock Solution I: NH4Cl 1 500 mg, MgCl2*6H2O 1 200 mg, CaCl2*2H2O 1 800 mg, MgSO4*7H2O 1 500 mg, KH2PO4 160 mg and H2O deionised ad 1 000 mL.
Stock Solution II: FeCl3*6H2O 64 mg, Na2EDTA*2H2O 100 mg and H2O deionised ad 1 000 mL.
Stock Solution III: H3BO3 185 mg, MnCl2*4H2O 415 mg, ZnCl2 3 mg, CoCl2*6H2O 1.5 mg, CuCl2*2H2O 0.01 mg, Na2MoO4*2H2O 7 mg and H2O deionised ad 1 000 mL.
Stock Solution IV: NaHCO3 50 g and H2O deionised ad 1 000 mL.
- Intervals of water quality measurement: pH was measured at the beginning and the end of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Light intensity: 5 000 lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Before the start of incubation and every 24 hours, the cell number was determined with an electronic particle counter.
- At the end of the test, the treatments were examined microscopically in order to assess the appearance of the algae and detect abnormalities (e.g. caused by the exposure to the test material).

TEST CONCENTRATIONS
- Test concentrations: Nominal: 0.032, 0.1, 0.32, 1, 3.2 and 10 mg/L.
- The concentrations to be tested were based on non GLP pre-tests.


CALCULATIONS
Growth Rate: The specific growth rate μ was calculated using the following equation:

µ = [ ln(Nn) – ln(N0) ] / tn

with:
µ = growth rate;
tn = time of the last measurement in days
N0 = nominal number of cells at time t0
Nn = nominal number of cells at time tn

Yield: The yield was calculated using the following equation:

Yield = N72 – N0

with:
N0 = number of cells at time t = 0 h
N72 = number of cells at time t = 72 h

Inhibition: All inhibition values were calculated by comparing the value for the endpoint of the treatment with the respective mean value of all blank controls (100 %). Inhibition values are given in percent.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence interval: 0.11-0.81 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.06 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % confidence interval: 0.04-0.09 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.01 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.:

0.04 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.04 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

ANALYTICAL DETERMINATION
The measured values are all very low and hardly detectable, except for the highest concentration. Therefore, no geometric mean could be calculated. Since the lower concentrations were prepared by dilution of the WAF, the real concentrations were calculated on the basis of the measured concentration in the WAF and the dilution factor.

VALIDITY
- Increase Factor: The cell concentration in the blank control should increase by a factor of at least 16 within 72h.
- Daily Growth Rates: Mean coefficient of variation of daily growth rates in the blank controls should be 35 % at the most. Coefficient of variation of average growth rate during the whole test period should be 7 % at the most.

Values and assessment: The daily growth rates of the blank controls were calculated. Means, standard deviations and coefficients of variation were determined:
- Increase factor biomass = 47: Valid
- Mean coefficient of variation of daily growth rates = 13 %: Valid
- Coefficient of variation of average growth rate during the whole test period = 1 %: Vaild

Results with reference substance (positive control):

- The study was performed under GLP conditions in January 2018 in a separate reference test.
- 72h ErC50: 0.79 mg/L (95 % CI: 0.79 mg/L – 0.79 mg/L)
- 72h EyC50: 0.39 mg/L (95 % CI: 0.35 mg/L – 0.45 mg/L)
- The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

Reported statistics and error estimates:

Calculation of results was performed with the help of validated software (Microsoft Excel®). The estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.2.0. All calculations are performed with unrounded values. Therefore re-calculation with rounded values may lead to slightly different results.

Table 1: Measured Concentrations IC and TC

Nominal Concentration Test Material (mg/L)	Measured TC t=0 h (mg/L)	Measured TC t=72 h (mg/L)	Measured IC t=0 h (mg/L)	Measured IC t=72 h (mg/L)
Blank Control	7.90	7.23	6.96	6.59
0.032	7.70	7.31	6.70	6.48
0.1	7.81	7.32	6.87	6.67
0.32	7.85	7.63	7.08	7.02
1	7.89	7.67	7.09	6.95
3.2	8.42	8.34	6.90	7.15
10	10.36	10.53	6.95	7.37

LOQ (Limit of quantification) TC = 3.54 mg/L

LOQ (Limit of quantification) IC = 1.79 mg/L

 

Table 2: Measured Concentrations DOC

Nominal Concentration Test Material (mg/L)	Measured DOC (TI-IC) t=0 h (mg/L)	Measured DOC (TI-IC) t=72 h (mg/L)	Measured DOC Minus Blank Control t=0 h (mg/L)	Measured DOC Minus Blank Control t=72 h (mg/L)
Blank Control	0.94	0.64	-	-
0.032	1.00	0.83	0.05	0.19
0.1	0.94	0.66	-0.01	0.02
0.32	0.76	0.61	-0.18	-0.03
1	0.80	0.72	-0.15	0.08
3.2	1.52	1.19	0.58	0.55
10	3.41	3.16	2.47	2.52

 

Table 3: Calculated Test Material Concentrations

Nominal Concentration Test Material (mg/L)	Calculated Concentration of Test Material t=0 h (mg/L)	Calculated Concentration of Test Material t=72 h (mg/L)
Blank Control	-	-
0.032	0.08	0.26
0.1	-0.01	0.02
0.32	-0.26	-0.04
1	-0.21	0.11
3.2	0.80	0.76
10	3.42	3.50

 

Table 4: Calculated Concentrations Based on the Analytically Determined Value for the WAF

Nominal Concentration (mg/L)	Calculated Concentrations (mg/L)
Blank Control	-
0.032	0.011
0.1	0.035
0.32	0.111
1	0.346
3.2	1.108
10	3.462

 

Table 5: Cell Numbers and Microscopical Observations

Nominal Concentration (mg/L)	Parameter	Cell Number/mL				Normal and Healthy Appearance of the Algae at End of Test
		0 h	24 h	48 h	72 h
Blank control	Mean	2440	7900	32887	115073	Yes
	SD	0	387	4392	5406
0.032	Mean	2440	8113	27273	117573	Yes
	SD	0	835	1070	3954
0.1	Mean	2440	7860	27453	96193	Yes
	SD	0	220	720	12565
0.32	Mean	2440	6650	17400	19550	Yes
	SD	0	14	877	2447
1	Mean	2440	6947	16653	18853	No cells visible
	SD	0	1260	2457	2120
3.2	Mean	2440	6400	5687	5653	No cells visible
	SD	0	779	541	1202
10	Mean	2440	4967	5120	5313	No cells visible
	SD	0	447	1413	597

SD = Standard Deviation

 

Table 6: Growth Rate μ and Yield Inhibition

Nominal Concentration (mg/L)	Parameter	Growth Rate (0-72 h) [day^-1]	Yield (0-72 h) [Cell Concentration/mL]	% Inhibition Growth Rate (0-72 h)	% Inhibition Yield (0-72 h)
Blank control	Mean	1.28	112633	0	0
	SD	0.02	5406
0.032	Mean	1.29	115133	-0.57	-2.22
	SD	0.01	3954
0.1	Mean	1.22	93753	4.78	16.76
	SD	0.05	12565
0.32	Mean	0.69	17110	46.09	84.81
	SD	0.04	2447
1	Mean	0.68	16413	47.04	85.43
	SD	0.04	2120
3.2	Mean	0.28	3213	78.56	97.15
	SD	0.07	1202
10	Mean	0.26	2873	79.91	97.45
	SD	0.04	597

SD = Standard Deviation

Negative inhibition values indicate a stimulation of algal growth compared to the blank control.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study the 72 hour EC50 values were 0.30 and 0.06 mg/L, for growth rate and yield, respectively and the 72 hour NOEC values were 0.01 and 0.04 mg/L for growth rate and yield, respectively.

Executive summary:

The toxicity of the test material to aquatic algae was investigated in accordance with the standardised guidelines OECD 201 and EU Method C.3., under GLP conditions using Desmodesmus subspicatus.

One valid experiment was performed.

The study was performed using 6 concentrations ranging from 0.032 to 10 mg/L. Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate μ and the yield were determined from the cell number at the respective observation times.

Significant inhibition of algal growth was observed at the following concentrations: 0.32 – 10 mg/L (Yield) and 0.1 – 10 mg/L (Growth Rate).

The water-accommodated fraction (WAF) was prepared for the test. This was done by mixing 10 mg/L with the corresponding amount of algal medium (demineralised water enriched with minerals but without algae) and shaking for 24 hours. The resulting solution was filtrated through 0.45 μm PTFE filters. Lower test concentrations were prepared by dilution of the WAF (10 mg/L) in algal medium. This was in accordance with OECD Guidance Document No. 23 where is stated that dilution of a WAF may be the only applicable method for chemicals being toxic at concentrations of 1 mg/L or lower.

At the start and at the end of the test, the content of the test item in the test solutions was estimated by determination of the dissolved organic carbon (DOC) content in the test solutions using a carbon analyser. The measured values are all very low and hardly detectable, except for the highest concentration. Therefore no geometric mean could be calculated for the lower concentrations. Since the lower concentrations were prepared by dilution of the WAF, the real concentrations were calculated on the basis of the geometric mean in the WAF and the dilution factor.

The 72h-EC50 values of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

Under the conditions of this study the 72 hour EC50 values were 0.30 and 0.06 mg/L, for growth rate and yield, respectively and the 72 hour NOEC values were 0.01 and 0.04 mg/L for growth rate and yield, respectively.

Description of key information

Under the conditions of this study the 72 hour EC50 values were 0.30 and 0.06 mg/L, for growth rate and yield, respectively and the 72 hour NOEC values were 0.01 and 0.04 mg/L for growth rate and yield, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.3 mg/L

EC10 or NOEC for freshwater algae:

0.01 mg/L

Additional information

The toxicity of the test material to aquatic algae was investigated in accordance with the standardised guidelines OECD 201 and EU Method C.3., under GLP conditions using Desmodesmus subspicatus. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

One valid experiment was performed.

The study was performed using 6 concentrations ranging from 0.032 to 10 mg/L. Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate μ and the yield were determined from the cell number at the respective observation times.

Significant inhibition of algal growth was observed at the following concentrations: 0.32 – 10 mg/L (Yield) and 0.1 – 10 mg/L (Growth Rate).

The water-accommodated fraction (WAF) was prepared for the test. This was done by mixing 10 mg/L with the corresponding amount of algal medium (demineralised water enriched with minerals but without algae) and shaking for 24 hours. The resulting solution was filtrated through 0.45 μm PTFE filters. Lower test concentrations were prepared by dilution of the WAF (10 mg/L) in algal medium. This was in accordance with OECD Guidance Document No. 23 where is stated that dilution of a WAF may be the only applicable method for chemicals being toxic at concentrations of 1 mg/L or lower.

At the start and at the end of the test, the content of the test item in the test solutions was estimated by determination of the dissolved organic carbon (DOC) content in the test solutions using a carbon analyser. The measured values are all very low and hardly detectable, except for the highest concentration. Therefore no geometric mean could be calculated for the lower concentrations. Since the lower concentrations were prepared by dilution of the WAF, the real concentrations were calculated on the basis of the geometric mean in the WAF and the dilution factor.

The 72h-EC50 values of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

Under the conditions of this study the 72 hour EC50 values were 0.30 and 0.06 mg/L, for growth rate and yield, respectively and the 72 hour NOEC values were 0.01 and 0.04 mg/L for growth rate and yield, respectively.