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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 - 14 Feb 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
28 Jul 2011
Deviations:
yes
Remarks:
(1) The pH (control) increased by 0.2 (8 - 8.2). Required: max. 1.5 units. (2) Light intensity was 40 instead of the recommended 60 - 120 µE*m^-2*s^-2 but enabled optimal growth to fulfill the OECD quality criteria.
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
24 Aug 2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Swiss Federal Office of Public Health, Bern, Switzerland (26 Jan 2015)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.100, 0.316, 1.00, 3.16, 10.0, and 31.6 mg/L (nominal). The LOQ is higher than the two lowest test concentrations (0.1 and 0.316 mg/L). This has no influence on the evaluation of the test.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test concentrations were prepared by dilution of a 31.6 mg/L stock solution in algal medium. The pH of the OECD medium was adjusted before inoculation with algal culture (8.0 ± 0.2) and then the test solutions were filter sterilized (0.45 µm cellulose acetate membrane sterility syringe filter, VWR, International)
- Controls: Blank controls containing test medium and algae (6 replicates).
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Green unicellular microalgae
- Source: Collection of Algal Culture, Institute of Freshwater Ecology, University of Göttingen, Germany
- Method of cultivation: 250 mL flask containing 100 mL sterile OECD medium inoculated with cell material from axenic slope cultures; Illumination: continuous (2000 - 3000 lux) from Osram Fluora L18W77 and Osram Daywhite L18W840 (Osram AG, Winterthur, Switzerland); Temperature: 22 ± 2 °C
- Other: The culture is re-purchased yearly to control sensitivity.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.5 ± 0.6 °C
pH:
blank: 8.0 (0 h), 8.2 (72 h)
highest concentration: 7.9 (0 h), 7.8 (72 h)
Nominal and measured concentrations:
control, 0.100, 0.316, 1.00, 3.16, 10.0, and 31.6 mg/L (nominal)
control, < LOQ,
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL all-glass flasks filled with 100 mL test medium.
- Renewal rate of test solution: No renewal.
- Initial cells density: 2 - 5E+03 cells/mL = 0.5 - 0.85 µg dry weight/mL (i.e. OD680 = ca. 0.005 units)
- Control end cells density: 0.568 units (OD680)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes (OECD medium)

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: Same as test.
- Intervals of water quality measurement: pH was measured at 0 and 72 h in the medium which is used for the control and in the saturated solutions.

OTHER TEST CONDITIONS
- Sterile test conditions: The test solutions were filter sterilized (0.45 µm cellulose acetate membrane sterile syringe filter, VWR, International).
- Adjustment of pH: The pH of the OECD medium was adjusted before inoculation with algal culture (pH 8.0 ± 0.2).
- Photoperiod: Continuous
- Light intensity and quality: ca. 3000 lux (40 µE*m^-2*s^-2) from Osram Fluora L18W77 and Osram Daywhite L18W840 (Osram AG, Winterthur, Switzerland). The light intensity is maintained within ± 6% of the average light intensity over the incubation area (required: ± 15%). The present light intensity is lower than the the 60 - 120 µE*m^-2*s^-2 recommended by the OECD testing guideline 202. However, based on the laboratory experience, the two lamps enable optimal growth conditions at a light intensity of 40 mE*m^-2*s^-2 to fulfill the required quality criteria according to OECD while yielding the "fittest" algal cells.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Spectrophotometer (wavelength: 680 nm) after 0, 24, 48 and 72 h.
- Other: Aliquots of 5 mL were removed from each test flask under sterile conditions. Cell concentrations at 0 h were only determined in the control vessels.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Range finding study: Yes, non-GLP.
- Test concentrations: 1, 10, and 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: Yes. In the range finding test inhibition of the average growth rate was 19, 60, and 102% at concentrations 1, 10, and 100 mg/L (nominal), respectively.
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.04 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 0.625 - 1.47 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
5.98 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 4.95 - 7.23 mg/L
Details on results:
- Exponential growth in the control: Yes, the biomass in the control cultures increased exponentially by a factor of > 16 within the 72 h test period corresponding to an average specific growth rate of 1.51 (required: > 0.92).
- Observation of abnormalities: No
- Effect concentrations exceeding solubility of substance in test medium: No
Reported statistics and error estimates:
The ErC50 and EyC50 values were calculated using ToxRat Standard Version 2.10 (ToxRat Solutions GmbH, Alsdorf, Germany). For the calculation of confidence intervals, values below 0% were set to 0.1% inhibition. Data were fit with the Logit regression model and the NOEC was determined by Dunnett´s test.

ANALYTICAL RESULTS

The LOQ of the method (0.5 mg/L) is higher than the two lowest test concentrations (0.100 and 0.316 mg/L). This has, however, no influence on the evaluation of the test.

HPLC measurements showed that the geometric mean measured test concentrations were within 91 - 105% of the nominal concentrations after 72 h, indicating that the test item was fully dissolved and stable for the duration of the test. Since test concentrations were maintained within ± 20% of the nominal concentrations throughout the test, effect concentrations were calculated based on nominal concentrations.

BIOLOGICAL RESULTS

The calculated median effect concentration ErC50 based on growth rate was 5.98 mg/L (95% CI: 4.95 - 7.23 mg/L), the ErC10 (72 h) was 1.04 mg/L (95% CI: 0.625 - 1.47 mg/L) and the NOErC (72 h) was 0.100 mg/L.

With respect to biomass production, the calculated EyC50 (72 h) was 1.28 mg/L (95% CI: 1.23 - 1.34 mg/L) and the NOEyC (72 h) was < 0.100 mg/L.

Table 1. Average specific growth rates between 0 and 72 h of exposure.

Nominal concentration [mg/L]

 

Replicate

 

GRaa)

µ3-0[d^-1]b)

 

GRaa)

µ3-0[d^-1]b)

mean values

 

Coefficient of variation [%]

 

Inhibition

[%]

 

Inhibition mean value [%]

 

Control

A

1.511

 

1.51

b)

 

0.1

c)

0.2

0.1

0.0

-0.1

0.0

-0.1

 

0.0

B

1.513

C

1.515

D

1.517

E

1.514

F

1.517

 

0.100

A

1.505

 

1.50

 

0.5

0.6

1.5

0.6

 

0.9

B

1.492

C

1.506

 

0.316

A

1.477

 

1.48

 

0.0

2.5

2.5

2.6

 

2.5

 

B

1.476

C

1.476

 

1.00

A

1.341

 

1.34

 

0.3

11.5

11.0

11.3

 

11.3

B

1.349

C

1.343

 

3.16

A

0.981

 

0.99

 

1.2

35.2

33.7

35.0

 

34.6

B

1.004

C

0.984

 

10.0

A

0.686

 

0.66

 

4.7

54.7

58.8

56.7

 

56.7

B

0.624

C

0.656

 

31.6

A

-0.061

 

-0.06

 

0.0

104.0

104.0

104.0

 

104.0

B

-0.061

C

-0.061

a)Average specific growth rate during the whole test period 0 – 3 d.

b)The biomass in the control cultures increased exponentially by a factor of > 16 within the 72 h test period corresponding to an average specific growth rate of 1.51 (required: > 0.92).

c)The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 0.1% (required: ≤ 7%).

Description of key information

ErC10 (72 h) = 1.04 mg/L (nominal, OECD 201, D. subspicatus)

ErC50 (72 h) = 5.98 mg/L (nominal, OECD 201, D. subspicatus)

Key value for chemical safety assessment

EC50 for freshwater algae:
5.98 mg/L
EC10 or NOEC for freshwater algae:
1.04 mg/L

Additional information

There is one key GLP study available investigating the toxic effects of the substance toward aquatic algae according to the OECD guideline 201.

In a static test, Desmodesmus subspicatus was exposed to the nominal test item concentrations of 0.1, 0.316, 1.0, 3.16, 10.0, and 31.6 mg/L under continuous light and controlled conditions for 72 h. A control was run in parallel. Since the test substance is soluble, the test solutions were prepared by the serial dilution of a 31.6 mg/L stock solution. The actual test concentrations were analytically verified by HPLC at the beginning (0 h), and after 24, 48 h and 72 h of exposure.

The geometric mean measured concentrations were 92 – 105% of the nominal concentration, showing that the test item was fully dissolved and stable throughout the duration of the test. Since the test concentrations were within ± 20% of the nominal concentrations, the effective concentrations (ErCx and EyCx) were calculated based on the nominal concentrations.

The calculated 72 h ErC10 was 1.04 mg/L (95% CI: 0.625 – 1.47 mg/L) and the ErC50 was 5.98 (95% CI: 4.95 – 7.23). The obtained NOErC (72 h) was 0.100 mg/L.