[No public or meaningful name is available]

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:

15 - 18 Mar 2016

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:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt Hildesheim, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control, 0.80, 2.53, 8.0, 25.3 and 80 mg/L
- Sampling method: Sampling was performed after 0 and 72 h.
- 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 of 80 mg/L was prepared with dilution water. The solution was stirred for 24 h at 1100 rpm at room temperature. The density of the substance was taken into account. Test solutions were prepared by stepwise dilution of the stock solution.
- Eluate: no
- Differential loading: no
- Controls: yes, test medium control
- Evidence of undissolved material: no, all test solutions were clear throughout of the exposure

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: SAG 61.81
- Source (laboratory, culture collection): Originally obtained from "Sammlung con Algenkulturen (SAG), Göttingen, Germany" and cultivated in teh laboratory. Fresh stocks are prepared every month on Z-agar (light intensity: 35-70 µE*m-2*s-1).
- Method of cultivation: Algae were cultivated in nutrient medium Z according to Lüttge et al. (1994).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol Ca+Mg/L

Test temperature:

21 - 22 °C

pH:

8.15 - 9.29 (control), 8.03 - 9.18 (0.713 mg/L), 8.06 - 9.13 (2.12 mg/L), 7.91 - 8.37 (7.07 mg/L), 7.87 - 8.02 (19.9 mg/L), 7.82 - 7.83 (69.8 mg/L)

Nominal and measured concentrations:

nominal: control, 0.80, 2.53, 8.0, 25.3 and 80 mg/L
measured: < LOQ, 0.713, 2.12, 7.07, 19.9 and 69.8 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile headspace flasks with a volume of 59 mL, flasks were sealed with aluminium tops with PFTE
- Type: closed
- Agitation: constantly shaken on a rotary shaker and oscillated at approx. 70 rpm
- Initial cells density: 6305 cells/mL
- Control end cells density: 59836 cells/mL (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, according to guideline

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Culture medium different from test medium: no, same as test
- Intervals of water quality measurement: The pH-value at the start of exposure was measured in one additional replicate of each test item concentration and the control. At the end of exposure, the pH-value was measured from pooled samples of each test item concentration and the control after measurement of the cell density. The room temperature was measured continuously. Light intensity was measured prior to the start of exposure.

OTHER TEST CONDITIONS
- Sterile test conditions: sterile test vessels were used
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 73.5 µE*m-2*s-1 (mean, n=24)

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: fluorescence of Chlorophyll a (excitation: 436 nm, emission: 685 nm); no self-fluorescence was found in a preliminary test at 80 mg/L.
- Other: Microscopic evaluation of the cells at the start and the end of exposure was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 0.31
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: conrol, 0.08, 0.80, 8.00, 80.0 mg/L
- Results used to determine the conditions for the definitive study: 0% growth inhibition at 0.08 mg/L, -1% growth inhibition at 0.80 mg/L, 29% growth inhibition at 8 mg/L, 100% growth inhibition at 80 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

7.07 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.96 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI

Remarks:

3.94 - 6.97 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI

Remarks:

7.26 - 13.6 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.713 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.713 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 0.713 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.67 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI

Remarks:

3.98 - 5.33 mg/L

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No morphological abnormalities were recorded.
- Any stimulation of growth found in any treatment: no
- Effect concentrations exceeding solubility of substance in test medium: All test solutions were clear and colorless.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: ErC50 (72 h) 0.564 mg/L (95% CI: 0.546 - 0.582 mg/L, with headspace), ErC50 (72 h) 1.15 mg/L (95% CI: 0.922 - 1.32 mg/L, without headspace); EyC50 (72 h) 0.322 mg/L (95% CI: 0.239 - 0.342 mg/L, with headspace), EyC50 (72 h) 0.509 mg/L (95% CI: 0.443 - 0.574 mg/L, without headspace)

Reported statistics and error estimates:

EC10-, EC20- and EC50-values with confidence intervals of growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression.
The NOEC / LOEC was determined by calculation of statistical significance of growth rate and yield. The following statistical tests were conducted:

- Shapiro-Wilk’s test on normal distribution was done with a significance level 0.01,
- Levene’s test on variance homogeneity was done with a significance level 0.01,
- William’s test on significant differences was done with a significance level 0.05.

Table 1: Growth inhibition of algae after 72 h

Geometric mean measured concentration [mg/L]	Replicate	Growth rate [d-1]	Inhibition of growth rate [%]
69.8	1	< LOQ	100
	2	< LOQ	100
	3	< LOQ	100
	Mean	n.a.	100
19.9	1	< LOQ	100
	2	< LOQ	100
	3	< LOQ	100
	Mean	n.a.	100
7.07	1	1.19	22
	2	1.18	22
	3	1.15	24
	Mean	1.17	23
2.12	1	1.41	7
	2	1.40	8
	3	1.41	7
	Mean	1.41	7
0.713	1	1.47	3
	2	1.48	2
	3	1.44	5
	Mean	1.46	4
Control	1	1.49
	2	1.49
	3	1.52
	4	1.52
	5	1.53
	6	1.54
	Mean	1.52

< LOQ = below the limit of quantification

Table 2: Measured concentrations at 0 and 72 h

Nominal test item concentration [mg/L]	0 h		72 h		Geometric mean [mg/L]
	Measured concentration [mg/L]	%	Measured concentration [mg/L]	%
80	66.8	84	72.9	91	69.8
25.3	19.8	78	20.0	79	19.9
8.0	7.55	94	6.62	83	7.07
2.53	2.29	91	1.97	78	2.12
0.80	0.757	96	0.663	83	0.713
Control	< LOQ		< LOQ		< LOQ

LOQ = 100 µg/L

Validity criteria fulfilled:

yes

Conclusions:

This study resulted in an ErC50 (72 h) of 8.28 mg/L and an ErC10 (72 h) of 5.96 mg/L (geom. mean measured concentration) based on inhibition of growth rate of P. subcapitata (OECD 201, GLP).

Executive summary:

The study was conducted according to OECD 201 and GLP under static conditions using Pseudokirchneriella subcapitata. The definitive test concentrations of 0.80, 2.53, 8.0, 25.3 and 80 mg/L were defined after a range-finding test with test concentration of 0.08, 0.80, 8.00, 80.0 mg/L. During the definitive test the exposure concentrations were verified by GC/MS analysis at the beginning and the end of exposure resulting in measured concentrations of 78 - 96% of nominal at the test start and 78 - 91% after 72 h. Inhibition of growth was recorded resulting in an ErC50 of 8.28 mg/L (95% CI 3.94 - 6.97 mg/L) and an ErC10 of 5.96 mg/L (95% CI 7.26 - 13.6 mg/L) after 72 h. All effect concentrations were based on the geometric mean measured concentration.

Description of key information

ErC50 (72 h): 8.28 mg/L based on growth inhibition of Pseudokirchneriella subcapitata (geometric mean measured concentration, OECD 201)

ErC10 (72 h): 5.96 mg/L based on growth inhibition of Pseudokirchneriella subcapitata (geometric mean measured concentration, OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:

8.28 mg/L

EC10 or NOEC for freshwater algae:

5.96 mg/L

Additional information

One experimental study is available investigating the toxicity of the substance to aquatic freshwater algae. The study was conducted according to OECD 201 and GLP under static conditions using Pseudokirchneriella subcapitata. The definitive test concentrations of 0.80, 2.53, 8.0, 25.3 and 80 mg/L were defined after a range-finding test with test concentration of 0.08, 0.80, 8.00, 80.0 mg/L. During the definitive test the exposure concentrations were verified by GC/MS analysis at the beginning and the end of exposure resulting in measured concentrations of 78 - 96% of nominal at the test start and 78 - 91% after 72 h. Inhibition of growth was recorded resulting in an ErC50 of 8.28 mg/L (95% CI 3.94 - 6.97 mg/L) and an ErC10 of 5.96 mg/L (95% CI 7.26 - 13.6 mg/L) after 72 h. All effect concentrations were based on the geometric mean measured concentration.