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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:
2003-05-19 to 2003-12-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Qualifier:
according to guideline
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations:1.0, 2.2, 4.6, 10, 22, 46 and 100 mg/L and blank control
- Sampling method: 0 h, 24 h and 72 h
10 mL from approximate centre of vessels
- Sample storage conditions before analysis: Not applicable, samples analysed on day of sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test solutions with a concentration of 1.0. 10 and 100 mg/L were prepared seperately. After a 10 minutes treatment with ultrasnic waves to accelerate the disolving of the test substance in the test medium, the test solutions were clear and colourless but the 10 and 100 mg/L concentrations contained a layer of foam. After the foam had disappeared from the highest test concentrations a ten-fold dilution was prepared from the 1.0 mg/L concentration. The final test solutions were clear and colourless
Final: Test concentrations prepared from 100 mg/L stock solution. 10 minutes magnetic stirring used to accelerate disolving of the test substance in the test media. Layer of foam dissolved in test medium by use of air pressure.
- Controls: Blank
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Thin foam on test concentrations in final test.
Test organisms (species):
Scenedesmus capricornutum
Details on test organisms:
TEST ORGANISM
- Common name: Blue/green algae
- Strain: NIVA CHL 1
- Source: In-house laboratory culture with known history
- Age at study initiation (mean and range, SD):
- Method of breeding: Inncoculation of growth medium with algal cells from a pure culture on agar. Suspensions continuously aerated and exposed to light at 23 ± 2°C
- Feeding during test: Not applicable

ACCLIMATION
- Acclimation period: 3 days (pre-culture)
- Acclimation conditions (same as test or not): Yes
- Type and amount of food: Medium M2 (ISO-standard)
- Feeding frequency: daily
- Health during acclimation (any mortality observed): not applicable, continuous culture
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
0.24 mmol (24 mg/L CaCO3)
Test temperature:
23 ± 2°C
pH:
8.3 ± 0.2
Dissolved oxygen:
Not measured
Salinity:
Not applicable
Nominal and measured concentrations:
1.0, 2.2, 4.6, 10, 22, 46 and 100 mg/L (nominal)
Average exposure concentrations: 0.36, 0.86, 1.7, 3.4, 7.2, 14 and 33 mg/L (see attached document 03-006F Exposure concentrations)

Analysis of test samples in the final test showed the test cncentrations were not stable especially over the last 48 h of the test period. The possible cause of loss of the test mateial was biodegradation. During the exposure period test solutions became gradually turbid probabaly due to bacterial growth.
The average exposure concentrations based on peak 1 and 2 were calculated and proved to be quite similar. Based on better analytical recoveries for peak 2, toxicological effects were related to the average expsure concentrations calculated for peak 2.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL glass
- Material, size, headspace, fill volume: 50 mL test solution + 1 mL algal suspension
- Aeration: Not specified
- Renewal rate of test solution (frequency/flow rate): Not renewed
- Initial cells density: 1E04 cells/mL
- Control end cells density: 1E04 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
Also 1 replicate of each test concentration without algae and 1 extra replicate of each test concentration and the blank-cintrol for sampling purposes.

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-Q water
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: No
- Photoperiod: constant
- Light intensity and quality: TLD Lamps of the type cool-white of 30 watt with light intensity 73 - 96 µE/m2/sec

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: microscope and a counting chamber and spectrophotometer
- Chlorophyll measurement: No

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 0.1 -100 mg/L increasing by a factor of 10
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate - periodical check on sensitivity of test system
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL = 0.69 - 5.2 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.45 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL = 0.15 - 1.29 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 0.36 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
6.8 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL = 3.2 - 14 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL = 0.68 - 3.1 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.86 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The test material reduced growth rates of Selenastrum capricornutum significantly at average exposure concentrations of 0.36 mg/L. upwards resulting in 99% inhibition at an average exposure concentration of 31 mg/L. Statistically signficant inhibition of cell growth was found at all average exposure concentrations tested (Bonferroni t and Tukey test).
Growth rates were in the range of the controls at the average exposure concentrations of 0.36 and 0.86 mg/L duringthe 72 h test period, whereas the growth rate of algae exposed to 1.7 mg/L and higher were increasingly reduced.
Although growth rate was reduced it increased as exposure progressed at average test substance concentrations of 1.7 up to 7.2 mg?l. Only the highest average test concentration (33 mg/L) growth rate decreased as exposure progressed. Statistically significant reduction of growth rate was found at average test concentrations of 1.7 mg/L and higher.
During the exposure period test solutions became gradually turbid, porbably due to baterial growth.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- ErC50: 0.89 mg/L (95% CL = 0.56 - 1.4 mg/L)
- Other: ISO standard ErC50 = 0.60 - 1.03 mg/L
Reported statistics and error estimates:
Analysis of test samples in the final test showed the test cncentrations were not stable especially over the last 48 h of the test period. The possible cause of loss of the test mateial was biodegradation. During the exposure period test solutions became gradually turbid probabaly due to bacterial growth.
The average exposure concentrations based on peak 1 and 2 were calculated and proved to be quite similar. Based on better analytical recoveries for peak 2, toxicological effects were related to the average expsure concentrations calculated for peak 2.

Table 1: % inhibition of cell growth during the final test

Average test material conc. (mg/L)

Cell growth (0-72 h)

Mean area (A)

Inhibition (%)

Blank control

1745.04

 

0.36

1460.92

16.3

0.86

1440.48

17.5

1.7

1104.92

36.7

3.4

571.64

67.2

7.2

89.60

94.9

14

114.04

93.5

33

15.88

99.1

 

Table 2: % reduction of growth rate at different time intervals

Average test material conc. (mg/L)

Mean growth rate

µ (0-24 h)

Reduction (%)

µ (0-48 h)

Reduction (%)

µ (0-72 h)

Reduction (%)

Blank control

0.04763

 

0.06644

 

0.06332

 

0.36

0.04356

8.6

0.06270

5.6

0.06079

4.0

0.86

0.03973

16.6

0.06172

7.1

0.06100

3.7

1.7

0.03156

33.7

0.05636

15.2

0.05750

9.2

3.4

0.01420

70.2

0.04272

35.7

0.04800

24.2

7.2

0.01134

76.2

0.01273

80.8

0.02486

60.7

14

0.03175

33.3

0.01373

79.3

0.02506

60.4

33

0.00944

80.2

0.00437

93.4

0.00259

95.9

Validity criteria fulfilled:
yes
Conclusions:
The test material reduced growth rates of Selenastrum capricornutum significantly at average exposure concentrations of 0.36 mg/L. upwards resulting in 99% inhibition at an average exposure concentration of 31 mg/L.
The ErC50 was 6.8 mg/L (95% confidence limits: 3.2 - 14 mg/L), the ErC10 was 1.5 mg/L (95% confidence limits: 0.68 - 3.1 mg/L) and the NOEC (growth rate) was 0.86 mg/L.
During the exposure period test solutions became gradually turbid, porbably due to baterial growth.
Executive summary:

In a 72 -hour algal inhibition study performed in accordance with OECD No 201, EU Method C3 and ISO Standard 8692 the test material was dosed at nominal concentrations of 1.0, 2.2, 4.6, 10, 22, 46 and 100 mg/L to Selenastrum capricornutum. The dose concentrations were chosen using a range finding study. A blank control was run in conjunction with the final test. Results of a positive control study on potassium dichromate were appended to the study to confirm sensitivity of the test system.

Analysis of the samples showed that the test material was unstable in the test medium after 48 hours and had decreased to below detection level after 72 hours. The Study Director indicated that loss was probably due to biodegradation of the test material. The average exposure concentrations were therefore 0.36, 0.86, 1.7, 3.4, 7.2, 14 and 33 mg/L.

The study met the acceptability criteria and was considered valid.

During the exposure period test solutions became gradually turbid probabaly due to bacterial growth.

The test material reduced growth rates of Selenastrum capricornutum significantly at average exposure concentrations of 0.36 mg/L. upwards resulting in 99% inhibition at an average exposure concentration of 31 mg/L.

The ErC50 was 6.8 mg/L (95% confidence limits: 3.2 - 14 mg/L), the ErC10 was 1.5 mg/L (95% confidence limits: 0.68 - 3.1 mg/L) and the NOEC (growth rate) was 0.86 mg/L.

Description of key information

Selenastrum capricornutum were exposed to the substance under static conditions for 72 hours in a GLP study performed according to OECD TG 201. The ErC50 (0 -72h) was 6.8 mg/L and the NOEC was 0.86 mg/L, both based on mean measured concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:
6.8 mg/L
EC10 or NOEC for freshwater algae:
0.86 mg/L

Additional information

One reliable study is available. In this study performed under GLP according to OECD TG 201 Selenastrum capricornutum (3 replicates/concentration) were exposed under static conditions to the substance for 72 hours at nominal concentrations of 10, 2.2, 4.6, 10, 22, 46 and 100 mg/L [Bouwman LM (2003b)]. The ErC50 (0 -72h) was 6.8 mg/L and the NOEC was 0.86 mg/L, both based on mean measured concentrations.