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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
Two deviations, see Principles of method if other than guideline. The study integrity was not adversely affected by the protocol deviations.
Principles of method if other than guideline:
There were two deviations from the protocol.
1. at the end of the range finding test at 10 mg/l samples were taken from the non-pooled solution in vessel 2 and from the pooled solutions from vessel 1 and 3 rather than from the pooled solutions of all three vessels. Evaluation: this yielded additional information that could be used for the evaluation of the results, since the growth in vessel 2 deviated from that is vessel 1 and 3.

2. In the final test, no samples were taken after 24h of exposure, even though the analytical results in the range finding test showed that the test conc. Was not stable throughout the test period. Evaluation: since measured concentrations were still well above the limit of detection, average exposure conc. could still be determined.

The study integrity was not adversely affected by the protocol deviations.
GLP compliance:
yes
Specific details on test material used for the study:
Clear colourless liquid, purity 96.5%.
Analytical monitoring:
yes
Remarks:
2ml samples were taken at the beginning and the end of the test and frozen until analysis. And the conc. of pentamethyl-trioxepane were determined in the samples with HPLC
Vehicle:
no
Details on test solutions:
Stock: pentamethyl-trioxepane 100 mg/l in test medium prepared after short stirring (10-17 min.)
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Selenastrum capricornutum, NIVA CHL 1. In house laboratory culture.

Algae stock solutions were started by inoculating growth M1 medium with algal cells from a pure culture on agar. Suspensions were aerated and exposed to light (60 to 120 μE/m2/s wavelength 400 to 700 nm) in a climate room (23 ± 2 °C). 3-4 days before the test 2.104 cells/ml were inoculated in M2 pre-culture medium and grown. Under the same conditions as used in the test. Cell density was measured immediately before use.
Test type:
static
Water media type:
other: Growth medium M1, test medium M2 according to ISO-standard. Dilution water source: Milli-Q water
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/l
Test temperature:
At the start of the test the test medium temp. was 23.5 °C. The incubator temp was maintained between 22.6 and 22.9 °C.
pH:
pH: 8.1-8.9
Nominal and measured concentrations:
Nominal concentrations:
Range finding test: 0.1, 1.0, 10 and 100 mg/l. Final test: 2.2, 4.6, 10, 22, 46 and 100 mg/l.

Measured concentrations:
Range finding test: only 10 and 100 mg/l, t=0 8.92 and 86.5 mg/l. t=72 5.00, 1.58 (this vessel was excluded from calc. because it contained more algae) and 68.5 mg/l.
Final test: At the end of the test the conc. declined > 80% therefore further calculations were performed with the average measured conc. Average: 1.6, 3.1, 3.7, 12, 33 and 73 mg/l. One sample was considered an outlier and excluded from further calculations.
Details on test conditions:
50 ml of stock solution was added to each replicate of a specific test and 1 ml of algal suspension was added providing a cell density of 104 cells/ml.
Range finding test: exponentially growing algal cultures were exposed to a range of 0.1 to 100 mg/l increasing by a factor 10 and to a blank-control in triplicate. One extra vessel without algae was used as a background to determine the algal cell density at each time interval.
Final test: exposure conc. pentamethyl-trioxepane; 2.2, 4.6, 10, 22, 46 and 100 mg/l in triplicate. 6 replicates of the blank-control and one vessel with the highest concentration of pentamethyl-trioxepane without algae.
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 95% CI from 4.1-18 mg/l
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
24 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI from 12-51 mg/l
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.8 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 95% CI from 0.86-3.9
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
4.7 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI from 2.2-10
Details on results:
In the range finding test one of three vessels was observed to contain more algae (79*104 cell/ml) after analysis this vessel was excluded from calculations. The expected EC50 for cell growth inhibition was close to the nominal conc. of 10 mg/l. The expected EC50 for growth rate inhibition was between 10 and 100 mg/l. The actual conc. of pentamethyl-trioxepane decreased more than 20% during the test. Final test: one sample with the lowest conc. was excluded form the calculations because the concentration seemed to have increased after 72h. All the other samples showed a decrease of more than 20%. The vessel containing 100 mg/l and no algae showed a greater decrease than the vessel with algae. Possibly the substance adsorbs to the glass vessel wall. The mean of the concentrations were used to calculate the average exposure conc.
Reported statistics and error estimates:
ANOVA, bonferroni t-test, TOXSTAT release 3.5, (1996, Gulley, boelter and bergman).

Final test: statistically significant inhibition of cell growth was found at test conc. of 3.1 mg/l and higher (Bonferroni t-test, α=0.05). statistically significant reduction of growth rate was found at the test conc. of 3.7 mg/l (Turkey test, α=0.05).
Validity criteria fulfilled:
yes
Remarks:
In the control, cell density increased with an average factor > 16 within 3 days and temp. and pH remained within ranges prescribed by the test guideline.
Conclusions:
Pentamethyl-trioxepane reduced growth rate of Selenastrum capricornutum significantly at 12 mg/l and higher.

The EC50 for cell growth inhibition (EBC50: 72h) was 8.5 mg/l with a 95% confidence interval ranging from 4.1 to 18 mg/l.
The EC10 for cell growth inhibition (EBC10: 72h) was 1.8 mg/l with a 95% confidence interval ranging from 0.86 to 3.9 mg/l.

The EC50 for growth rate reduction (ERC50: 72h) was 24 mg/l with a 95% confidence interval ranging from 12 to 51 mg/l.
The EC10 for growth rate reduction (ERC10: 72h) was 4.7 mg/l with a 95% confidence interval ranging from 2.2 to 10 mg/l.

The NOEC for cell growth inhibition was 1.6 mg/l, while the NOEC for growth rate reduction was 3.1 mg/l.
Executive summary:

The EC50 for cell growth inhibition (EBC50: 72h) was 8.5 mg/l with a 95% confidence interval ranging from 4.1 to 18 mg/l.

The EC10 for cell growth inhibition (EBC10: 72h) was 1.8 mg/l with a 95% confidence interval ranging from 0.86 to 3.9 mg/l.

The EC50 for growth rate reduction (ERC50: 72h) was 24 mg/l with a 95% confidence interval ranging from 12 to 51 mg/l.

The EC10 for growth rate reduction (ERC10: 72h) was 4.7 mg/l with a 95% confidence interval ranging from 2.2 to 10 mg/l.

The NOEC for cell growth inhibition was 1.6 mg/l, while the NOEC for growth rate reduction was 3.1 mg/l.

Description of key information

The EC50 for cell growth inhibition (EBC50: 72h) was 8.5 mg/l with a 95% confidence interval ranging from 4.1 to 18 mg/l.

The EC10 for cell growth inhibition (EBC10: 72h) was 1.8 mg/l with a 95% confidence interval ranging from 0.86 to 3.9 mg/l.

The EC50 for growth rate reduction (ERC50: 72h) was 24 mg/l with a 95% confidence interval ranging from 12 to 51 mg/l.

The EC10 for growth rate reduction (ERC10: 72h) was 4.7 mg/l with a 95% confidence interval ranging from 2.2 to 10 mg/l.

The NOEC for cell growth inhibition was 1.6 mg/l, while the NOEC for growth rate reduction was 3.1 mg/l.

Key value for chemical safety assessment

EC50 for freshwater algae:
24 mg/L
EC10 or NOEC for freshwater algae:
4.7 mg/L

Additional information