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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:
12 August 2013 - 15 August 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test method according to EC method C.3 and OECD 201. GLP study.
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100, 31.3, 9.8, 3.1 and 1.0 mg/L
- Sampling method: Analytical measurements were carried out daily at each test concentration and at the control at the start and at the end of the test.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Test concentrations were prepared with test item and test medium (OECD medium) according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23. prior to treatment. The test item is a UVCB material, a complex multi-component substance, which is only partially soluble in water. According to OECD No. 23., the toxicity of such substances were determined by preparing water-accommodated fractions (WAFs). Saturated test item solutions (nominal loading rate of 100, 31.3 and 9.8 mg/L) were prepared by dispersing/dissolving the appropriate amount of test item into the test medium (ISO medium) two days before treatment. These solutions were shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at approximately 20°C. A short settling time was considered to be inappropriate as the liquid phases did not separate visibly in the period after mixing. Hence a period of approximately 24 hour time was considered to be the most practicable settling time. After settling, centrifugation was found not to work for aiding separation (density is near that of water); filtration through a fine (0.22 μm) filter was found to be useful to prepare a suitable media for the study. Nominal test concentrations of 3.1 and 1.0 mg/L were prepared by appropriate dilution of nominal test concentrations of 9.8 mg/L and 3.1 mg/L respectively, since production of such low concentrations is impractical (as described in OECD 23).
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source (laboratory, culture collection): The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Germany
- Method of cultivation: Cultured under standardised conditions (OECD 201) in the Ecotoxicological Laboratory of CiToxLAB Hungary Ltd.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.9 – 23.2 °C
pH:
7.71 – 8.91
Nominal and measured concentrations:
Nominal concentrations: 1.0; 3.1; 9.8; 31.3 and 100 mg/L
Measured concentrations: See "Any other information on results incl. tables".
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): covered with air-permeable stoppers.
- Material, size, headspace, fill volume: 100 mL (observation flasks), 160 mL (analytical sampling).
- Initial cells density: 10E+04 algal cells per mL
- No. of vessels per concentration (replicates): 5 (3 for observations and 2 replicates for analytical measurements)
- No. of vessels per control (replicates): 6

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

TEST MEDIUM / WATER PARAMETERS
Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range-finding and definitive tests.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Continuously illuminated
- Light intensity and quality: 8117 lux (equivalent to 110 μE/m2/s), with fluorescent lamps (with a spectral range of 400-700 nm).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
The cell numbers were determined at 24, 48 and 72 hours after the treatment by manual cell counting using a microscopic method with a counting chamber. Microscopic observation of the algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 0 (control), 0.1, 10 and 100 mg/L loading rates (2 replicates per dose, 3 replicates per control)
- Results used to determine the conditions for the definitive study: 0.0, 0.3, 1.3, 13.0 and 29.9 % of growth inhibition at 0 (control), 0.1, 10 and 100 mg/L loading rates respectively.
Reference substance (positive control):
not required
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
5.82 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 4.91 – 6.91 mg/L
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
1.34 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 1.00 – 1.78 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Morphological deviations of the algal cells were observed by using a microscopic method with a counting chamber. At 48 h in the case of nominal loading rates of 3.1; 9.8 and 31.3 mg/L chuff cells were observed, and deformity of the algae cells was observed at nominal loading rate of 100 mg/L. At 72 h chuff cells were observed in the case of nominal loading rate of 3.1 mg/L and deformity of the algae cells was observed at nominal loading rates of 9.8; 31.3 and 100 mg/L.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Droplets resulting in physical effects were not observed in the test media.
Results with reference substance (positive control):
For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate is tested at least twice a year by the laboratory to demonstrate satisfactory test conditions. The date of the last study (Study Code: 13/019-022AL) with the reference item Potassium dichromate is (Batch Number: 0769128): 05 - 08 February 2013.
The 72h ErC 50 : 0.87 mg/L, (95 % confidence limits: 0.79 – 0.96 mg/L).
The 72h EbC 50 : 0.48 mg/L, (95 % confidence limits: 0.44 – 0.53 mg/L).
The 72h EyC 50 : 0.45 mg/L, (95 % confidence limits: 0.41 – 0.50 mg/L).
Reported statistics and error estimates:
The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3) and to demonstrate exponential growth for the entire study period. The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel for Windows software. The ErC50, EbC50 and EyC50 values of the test item were determined using Probit analysis by TOXSTAT software. Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software. For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test.

RESULTS:

Nominal loading rate (mg/L)

0-72 h

Growth rate (µ) and % inhibition of µ

Area under the Growth Curves (A) and Percentage Inhibition of A

Yield (Y) and % inhibition of Y

µ

%

A

%

Y

%

0

0.0589

0.0

1364.0

0.0

68.7

0.0

1.0

0.0580

1.6

1264.0

7.3

64.0*

6.8

3.1

0.0406*

31.1

460.0*

66.3

17.7*

74.3

9.8

0.0115*

80.5

72.0*

94.7

1.3*

98.1

31.3

0.0096*

83.7

60.0*

95.6

1.0*

98.5

100

0.0000*

100.0

0.0*

100.0

0.0*

100.0

* : statistically significantly different compared to the control values (Bonferroni t-Test; α = 0.05)

Parameter (0-72h)

Growth rate (mg/L)

Yield (mg/L)

Biomass (mg/L)

EL50 (95% CL)

5.82 (4.91-6.91)

2.31 (2.03-2.64)

2.72 (2.31-3.19)

EL10(95% CL)

1.34 (1.00-1.78)

0.95 (0.77-1.18)

0.81 (0.62-1.07)

EL20 (95% CL)

2.22 (1.76-2.80)

1.29 (1.08-1.54)

1.23 (0.98-1.54)

NOELR

1.0

<1.0

1.0

LOELR

3.1

1.0

3.1

Analytical results:

Chemical analysis of the media for the 3 main components of the UVBC test item identified measured geometric mean test item concentrations of 0.032; 0.053; 0.174; 0.232 and 0.255 mg/L at the nominal loading rates of 1.0; 3.1; 9.8; 31.3 and 100 mg/L respectively. It is concluded that the WAF method used was the most appropriate for ensuring exposure of organisms to water-soluble fractions of the UVCB test item. This procedure maximised exposure and is suitable for risk assessment. The analytical method was designed to measure the presence of the main components of the UVCB test item; however the WAF method is designed to test any extractable components that may enter the aquatic environment. Hence the results of the study are expressed in units of nominal test item concentrations, the analytical data provide evidence of exposure and supplementary information about the main components of the test item.

Validity criteria fulfilled:
yes
Remarks:
(cell density in control increased by a factor of 69.67 (>16%), mean coefficient variation for section-by-section specific growth rates in control was 15.50% (<35%), coefficient of variation of average specific growth rates in control was 0.59% (<7%).
Conclusions:
The 72h-EL50 of the test item was determined to be 5.82 mg/L loading rate (basis for effect: growth rate) in algae. The 72h-EL10 was 1.34 mg/L and 72h-NOELR was 1 mg/L.
Executive summary:

The effect of the test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subpicata according to EC method C.3 and OECD 201. Based on preliminary range-finding results, green algae were exposed to 0 (control), 1.0, 3.1, 9.8, 31.3 and 100 mg/L over a period of 72 hours under static conditions. The test design included five replicates (three for observations and two replicates for analytical measurements) at test concentrations and six replicates for the untreated control. The test item is a UVCB material, a complex multi-component substance, which is only partially soluble in water. According to OECD No. 23., the toxicity of such substances can be determined by preparing water-accommodated fractions (WAFs). Test concentrations were analytically determined at the beginning of the test and 24-hour intervals thereafter during the experiment. All validity criteria were met during this study. Morphological deviations of the algal cells were observed by using a microscopic method with a counting chamber. At 48 h in the case of nominal loading rates of 3.1; 9.8 and 31.3 mg/L chuff cells were observed, and deformity of the algae cells was observed at nominal loading rate of 100 mg/L. At 72 h chuff cells were observed in the case of nominal loading rate of 3.1 mg/L and deformity of the algae cells was observed at nominal loading rates of 9.8; 31.3 and 100 mg/L. Droplets resulting in physical effects were not observed in the test media. Regarding the biological effects, the 72h-EL50 of the substance was determined to be 5.82 mg/L loading rate (basis for effect: growth rate) in algae. The 72h-EL10 was 1.34 mg/L and 72h-NOELR was 1 mg/L.

Description of key information

Key study: Test method EC method C.3, OECD 201. GLP study: The 72h-EL50 of CNSL was determined to be 5.82  mg/L loading rate (basis for effect: growth rate) in green algae. The 72h-EL10 was 1.34 mg/L and 72h-NOELR was 1 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
5.82 mg/L
EC10 or NOEC for freshwater algae:
1.34 mg/L

Additional information

Key study: The effect of the test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata, over an exposure period of 72 hours with nominal loading rates of 1.0; 3.1; 9.8; 31.3 and 100 mg/L (based on preliminary range-finding results) produced by the WAF method. Test method was according to EC method C3 and OECD 201. At 48 h in the case of nominal loading rates of 3.1; 9.8 and 31.3 mg/L chuff cells were observed, and deformity of the algae cells was observed at nominal loading rate of 100 mg/L. At 72 h chuff cells were observed in the case of nominal loading rate of 3.1 mg/L and deformity of the algae cells was observed at nominal loading rates of 9.8; 31.3 and 100 mg/L. Droplets resulting in physical effects were not observed in the test media. Regarding the biological effects, the 72h-EL50 of the substance was determined to be 5.82 mg/L loading rate (basis for effect: growth rate) in algae. The 72h-EL10 was 1.34 mg/L and 72h-NOELR was 1 mg/L.