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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:

10 - 13 Apr 2017

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt Hildesheim, Germany (03.01.2017)

Analytical monitoring:

yes

Remarks:

GC-MS

Details on sampling:

- Concentrations: All concentration levels and the control at the start of exposure (0 h), after 24 h, 48 h and at the end of the exposure.
- Sampling method: Separate replicates with algae were prepared for each concentration level and the control for test item analysis at the beginning of the exposure and analysis after 24, 48, and 72 h. Additionally, samples without algae were prepared for the determination of the adsorption to aglae and glass at the end of the exposure. The adsorption to glass was analytically verified from the highest concentration level.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until the start of the analysis, if necessary. Prepared samples were stored in the autosampler at room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution with a nominal loading of 10 mg/L test item was prepared once with dilution water and stirred (1100 rpm) for 3 h at room temperature. The density of the test item was taken into account. After a separation phase of 10 min the stock solution was removed from the homogenous phase, from the bottom of the glass and was used for testing. The stock solution was checked after stirring via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion), which was negative. Five dilution levels were prepared from the stock solution.
- Controls: Dilution water without test item incubated under the same conditions as the test concentrations.
- Evidence of undissolved material: The test media were clear throughout the exposure phase.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular green algae
- Strain: SAG 61.81
- Age of inoculum (at test initiation): A 3 d old preculture, prepared in dilution water, was used as inoculum.
- Method of cultivation: Fresh stocks are prepared every month on Z-agar and kept at 2567 - 5130 lux for 24 h/d at the testing facility. Culture medium: Nutrient medium Z, according to Lüttge et al. 1994.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.8 °C

pH:

Control: 7.97 (0 h), 9.34 (72 h)
Treatments: 7.92 - 8.09 (0 h), 8.34 - 9.38 (72 h)

Nominal and measured concentrations:

Control, 0.0316, 0.10, 0.316, 1.00, 3.16, and 10.0 mg/L (nominal)
< LOQ , 0.00717, 0.0175, 0.0868, 0.616, 1.16, and 7.11 mg/L (time weighted average measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: 59 mL sterile flasks with aluminium tops with PTFE seals, filled with 59 mL test volume (no headspace).
- Type: Closed
- Initial cells density: 6839 cells/mL
- Control end cells density: 1538744 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes, OECD 201 medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to the guidelines
- Culture medium different from test medium: The preculture was cultured in the dilution water used for the test.
- Intervals of water quality measurement: Room temperature was measured continuously. Light intensity was measured at the start of exposure. The pH values at the start of the exposure were measured in one additional replicate of each test item concentration and the control. At the end of exposure, the pH values were measured from pooled samples of each test item concentration and the control.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Photoperiod: 24 h light/d
- Light intensity and quality: 5938 lux (mean, n = 32)
- Incubation: The test flasks were placed on a rotary shaker and oscillated at approximately 70 rpm. Flasks were positioned randomly and repositioned daily.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Chlorophyll-a fluorescence was measured with a Fluorometer at an excitation wavelength of 436 nm and an emission wavelength of 685 nm (Microplate Reader Chameleon V)
- Other: Dilution water was used as background signal and no auto-fluorescence was detected in the saturated solution in the 1st preliminary range-finding test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10^(1/2)
- Range finding study: Two non-GLP range-finding tests were conducted.
- Test concentrations: 1st range-finding test: 100, 10, and 1% of saturated solution with a nominal loading level of 1 mL/L; 2nd range-finding test: 2.5 and 10.0 mg test item/L (nominal loading)
- Results used to determine the conditions for the definitive study: Yes, the dilution levels of the stock solution used in the definitive test were based on the results of the preliminary range-finding tests.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.178 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval

Remarks:

0.151 - 0.216 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.052 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval

Remarks:

0.0403 - 0.0661 mg/L

Details on results:

- Exponential growth in the control: Yes
- Observation of abnormalities: No morphological abnormalities were observed
- Any observations that might cause a difference between measured and nominal values: The test media were clear throughout the exposure phase.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes, the toxicity of the reference substance is in the valid range of the test facility SOPs.
- ErC50 (72 h): 0.435 mg/L (with headspace) and 0.811 mg/L (without headspace)
- Other: The most recent test with the reference item dates from 11 - 14 Oct 2016 (with headspace) and 10 - 13 Oct 2016 (without headspace).

Reported statistics and error estimates:

The NOEC / LOEC were determined by calculation of the statistical significance of growth rate and yield with the following tests. The Shapiro-Wilk´s test on normal distribution was done with a significance level of 0.01 for growth rate and yield. Levene's test on variance homogeneity was done with a significance level of 0.01 for growth rate and yield. Multiple Sequentially-rejective Welsh-t-test was performed with a significance level of 0.05 for growth rate and yield. Calculations were carried out with Excel (Microsoft Corporation), SigmaPlot (SPSS Inc.), GraphPad Prism (GraphPad Software, Inc.), and ToxRat Professional V3.2.1 (ToxRat Solutions GmbH).

VALIDITY CRITERIA

The study meets the validity criteria of the guideline (Table 1).

 

Table 1. Validity criteria.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	225-fold increase (specific growth rate 1.80/d)	Yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	The section-by-section specific growth rate was 23.8%	Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus.	The coefficient of variation was 2.53%	Yes

 

ANALYTICAL RESULTS

At the start of exposure, the measured test item concentrations ranged from 84 – 90% of the nominal values. After 24 and 48 h the measured concentrations dropped to 44 – 83% and < LOQ – 87%, respectively. At the end of exposure, the measured concentration was between < LOQ and 43% of the nominal concentration (Table 2). Therefore, all effect values were reported as time weighted average test item concentrations.

Adsorption to algae and test vessels (glass) was also tested. No significant concentrations were detected on neither of the surfaces (glass and algae).

 

Table 2. Measured concentrations and percentage of the nominal concentrations of the test item.

Nominal test item concentration [mg/L]	Test item								Time weighted average [mg/L]
	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]
10.0	8.82	88	7.58	76	8.71	87	3.37^3)	34	7.11
3.16	2.79	88	2.63^2)	83	1.65	52	0.0450^4)	1.4	1.16
1.00	0.896	90	0.655	65	0.576	58	0.430	43	0.616
0.316	0.283	90	0.198	63	0.0668^3)	21	0.00862	2.7	0.0868
0.10	0.0877	88	0.0435	44	0.00832^3)	8.3	< LOQ^1)		0.0175
0.0316	0.0267^5)	84	0.0180	57	< LOQ^1)		< LOQ^1)		0.00717
Control	< LOQ		< LOQ		< LOQ		< LOQ		< LOQ

Meas. conc. = Measured concentration of the test item (dilution and enrichment factor taken into account)

LOQ = Limit of quantification (0.005 mg test item/L)

1) = ½ LOQ taken into account for calculation of the time weighted average

2) = Injection of the freshly diluted extract resulting from first sample preparation on 2017-04-12

3) = Mean value of two injections with different dilution factors

4) = Mean value of two injections on 2017-04-13 and two re-analyses on 2017-04-18

5) = Measured on 2017-04-11

BIOLOGICAL RESULTS

The substance was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours (Table 3). The EC50 values with 95% confidence intervals for inhibition of growth rate (ErC50) was 0.0575 (0.0403 – 0.0727) mg/L. The EC10 value with 95% confidence intervals for inhibition of growth rate (ErC10) was 0.0153 mg/L (< 0.00717 – 0.0289 mg/L). The NOEC-values for inhibition of growth rate and yield after 72 hours were 0.0868 and 0.0175 mg/L, respectively.

Table 3. Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Time weighted average test item concentration [mg/L9	Replicate No.	Growth rate [d^-1]	Inhibition of growth rate [%]	Yield [cells/mL]	Inhibition of yield [%]
7.11	1	n.a.	100	n.a.	100
	2	n.a.	100	n.a.	100
	3	n.a.	100	n.a.	100
	Mean	n.a.	100	n.a.	100
1.16	1	n.a.	100	n.a.	100
	2	n.a.	100	n.a.	100
	3	n.a.	100	n.a.	100
	Mean	n.a.	100	n.a.	100
0.616	1	0.106	94	2557	100
	2	0.202	89	5685	100
	3	0.192	89	5334	100
	Mean	(+) 0.167	91	(+) 4525	100
0.0868	1	1.54	15	681629	56
	2	1.50	17	606899	60
	3	1.22	33	255370	83
	Mean	(-) 1.42	21	(+) 514633	66
0.0175	1	1.77	2	1386912	9
	2	1.78	1	1431387	7
	3	1.73	4	1204915	21
	Mean	(-) 1.76	2	(-) 1341071	12
0.00717	1	1.84	-2	1676586	-9
	2	1.77	2	1381645	10
	3	1.78	1	1403298	8
	Mean	(-) 1.79	0	(-) 1487176	3
Control	1	1.84		1677757
	2	1.83		1662073
	3	1.83		1661956
	4	1.82		1589274
	5	1.78		1415236
	6	1.72		1185135
	Mean	1.80		1531905

negative values = growth stimulation

n.a. = not applicable

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Conclusions:

Based on the outcome of the growth inhibition test, an ErC50 (72 h) of 0.178 mg/L (TWA) and an ErC10 (72 h) of 0.0525 mg/L (TWA) was determined (OECD 201, P. subcapitata).

Description of key information

ErC10 (72 h) = 0.0525 mg/L (time weighted average, OECD 201, P. subcapitata)

ErC50 (72 h) = 0.178 mg/L (time weighted average, OECD 201, P. subcapitata)

Key value for chemical safety assessment

EC50 for freshwater algae:

0.178 mg/L

EC10 or NOEC for freshwater algae:

0.052 mg/L

Additional information

There is one study available, in which the toxicity of the substance toward aquatic algae was investigated according to OECD guideline 201 and GLP.

In a static test, Pseudokirchneriella subcapitata at an initial cell density of 6839 cells/mL was exposed to five test item concentrations ranging from 0.0316 – 10.0 mg/L in a geometric series with a dilution factor of 10^(1/2) for 72 h. Test item concentrations were analytically verified by GC-MS at the start of the test (0 h) and every 24 h until test end.

The measured test item concentrations at the start of exposure ranged from 84 – 90% of the nominal values. After 24 and 48 h the measured concentrations dropped to 44 – 83% and < LOQ – 87%, respectively. At the end of exposure, the measured concentrations were between < LOQ and 43% of nominal. No significant adsorption of the test item to glass and algae was detected. All effect values were reported as time weighted average test item concentrations.

After 72 h, the substance was found to inhibit the growth of the freshwater green alga. The determined ErC50 (72 h) was 0.178 mg/L and the ErC10 (72 h) was 0.0525 mg/L.