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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:
05.01. - 25.01.2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Freshwater Algae and Cyanobacteria, Growth Inhibition Test, Commission Regulation (EC) No. 266/2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
at the beginning of the test and at the end of the test in all test concentrations
- Sampling method:
The analytical determination of the test item concentration was performed at the beginning and at the end of the test. The samples for analysis were taken from the highest (128 mg·L-1), the lowest (5 mg·L-1) test concentration and from the highest (128 mg·L-1) concentration without algae. The samples for analysis (0 hours) were prepared at the beginning of the test and immediately delivered in transport box to analytical laboratory.
The samples for analysis at the end of the test (72 hours) were delivered to analytical laboratory immediately after the end of testing.

- Sample storage conditions before analysis: The samples were analyzed on the day of delivery. All samples were stored at laboratory temperature.

The quantification of 5-aminotetrazole in samples taken from the test substance solutions used in the Alga, Growth Inhibition Test was performed by HPLC method developed and validated at GLP test facitility.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The stock solution of the test substance was prepared in the test medium. 50 mg of the test substance was weighed into 500 mL of the test medium for the preliminary test and 100 mg of the test substance was weighed into 500 mL of the test medium for the full test.

- Differential loading: The test was performed in a range nominal concentrations from 5 mg·L-1 to 128 mg·L-1. For the test was used geometric concentration series of the test item with factor 1.7.

- Controls: The test contained 3 parallel series of the test substance concentrations and 3 controls without the test substance.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
Test medium - the water with conductivity smaller than 5 μS·cm-1 was used for the preparation of solutions.

- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)):
The volume of the test solution was 50 mL in each testing flask.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: freshwater algae Desmodesmus subspicatus
- Strain: Desmodesmus subspicatus Brinkmann 1953/SAG 86.81
- Source: from the collection of autotrophic organisms of The Botanic Institute of the Czech Academy of Science, Třeboň on date 25.5.2017
- Method of cultivation: The strain culture was preinoculated from the stock culture and cultivated in flasks with the test medium on indirect daylight at laboratory temperature. Algae inoculum for the test was sampled from exponentially growing inoculum culture.
- Age of inoculum (at test initiation): The strain culture was always set to pre-culturing of cells for 3-4 days before the start of the test. Inoculum culture was kept 3-4 days under conditions at which the test was performed.

ACCLIMATION
- Acclimation period: 3-4 days
- Culturing media and conditions (same as test or not): same as test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23 – 23.5 °C
pH:
8.1
Conductivity:
1.13 µS·cm-1
Nominal and measured concentrations:
128, 75, 44, 26, 15, 9 and 5 mg·L-1
measured 5.25 mg.L-1 (0 h), 5.17 mg.L-1 (72 h)
measured 133.7 mg.L-1 (0 h), 135.2 mg.L-1 (72 h)
measured 132.3 mg.L-1 (0 h), 133.0 mg.L-1 (72 h) without algae
Details on test conditions:
Preliminary test
Stock solution of the test item: 50.05 mg / 500 mL-1
Test concentrations: 100, 50, 10, 5 and 1 mg·L-1
Conductivity of deionized water: 1.86 µS·cm-1
pH of the test medium: 8.0
Volume of inoculated algae culture: 1.48 mL in 200 mL of mixture
Lighting during the test: 7 050 – 7 090 lux
Temperature during the test: 22.5 – 23.0°C
Inicial density: 5000 cells per 1ml

Full test
Stock solution of the test item: 100.02 mg / 500 mL-1
Test concentrations: 128, 75, 44, 26, 15, 9 and 5 mg·L-1
Conductivity of deionized water: 1.13 µS·cm-1
pH of the test medium: 8.1
Volume of inoculated algae culture: 0.88 mL in 200 mL of mixture
Lighting during the test: 7 020 – 7 025 lux
Temperature during the test: 23.0 – 23.5°C
Inicial density: 5000 cells per 1ml

The test contained three parallel series of the test item concentration and three controls without the test item. The volume of the test solution was 50 mL in each testing flask.
The flasks were placed on a shaker under the lighting ramp and were incubated under continuous illumination and shaking for 72 hours. At the beginning and the end of the test the pH of the test mixtures was measured. The light intensity and temperature were measured every 24 hours. The density of algae culture was evaluated microscopically at 24, 48 and 72 hours. The cell density was measured by direct counting of living cells in Burker´s counting chamber.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
98.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The test quality criteria were fulfilled.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
The value EC50 for inhibition of growth rate (72h – ErC50) obtained from our last reference test meets the calculated range from the interlaboratory test.
The sensitivity of test species and proficiency of our laboratory in test performance was proved.

Reference test: 72 hour – ErC50 = 1.09 mg·L-1 (95% confidence limit: 0.91 – 1.30 mg·L-1)
Interlaboratory test: 72 hour – ErC50 = 0.51 – 1.19 mg·L-1
Validity criteria fulfilled:
yes
Conclusions:
The value of ErC50, EyC50, ErC10, EyC10 and the confidence intervals were calculated using the statistical software ToxRat Professional Version 3.2.1.
The determination of NOEC values were done by statistical software ToxRat Professional Version 3.2.1.
The nominal concentrations were used for all evaluations and results.
72 hour – ErC50 = 98.6 mg·L-1 (nominal concentrations)       (95% confidence limit: 72.6 – 132.2 mg·L-1)
72 hour – NOECr ˂ 5 mg·L-1 (nominal concentrations)
Executive summary:

The test item, 5-aminotetrazole, was tested for growth inhibition on algae Desmodesmus subspicatus.

The test was performed according to method C.3. - Freshwater Algae and Cyanobacteria, Growth Inhibition Test, Commission Regulation (EU) No. 2016/266.

The preliminary test was performed in a range of the test item nominal concentrations 1 – 100 mg·L-1.

The analytical results showed, that the test item 5-aminotetrazole was sufficiently stable in the test medium at conditions of the test in the concentration range 1 – 100 mg·L-1 .

Based on toxicity of the test item found in preliminary test, the full test was performed in a range of the test item nominal concentrations 5 – 128 mg·L-1.

The guideline specify, that if evidence is available to demonstrate that the concentration of the test item in the full test has been satisfactorily maintained within ± 20 per cent of the nominal or measured initial concentration throughout the full test, then the results can be based on nominal or measured initial values, which is the case of this study. The nominal concentrations were used for all evaluations and results.

 

Test results:

72 hour – ErC50 = 98.6 mg·L-1       (nominal concentrations)       (95% confidence limit: 72.6 – 132.2 mg·L-1)

72 hour – EyC50 = 17.7 mg·L-1       (nominal concentrations)       (95% confidence limit: 11.7 – 27.2 mg·L-1)

72 hour – ErC10 = 17.9 mg·L-1       (nominal concentrations)       (95% confidence limit: 14.1 – 22.7 mg·L-1)

72 hour – EyC10 = 3.3 mg·L-1        (nominal concentrations)       (95% confidence limit: 2.3 – 4.8 mg·L-1)

72 hour – NOECr ˂ 5 mg·L-1       (nominal concentrations)

72 hour – NOECy ˂ 5 mg·L-1       (nominal concentrations)

Description of key information

The value of ErC50, EyC50, ErC10, EyC10 and the confidence intervals were calculated using the statistical software ToxRat Professional Version 3.2.1.

The determination of NOEC values were done by statistical software ToxRat Professional Version 3.2.1. 

The nominal concentrations were used for all evaluations and results.

72 hour – ErC50 = 98.6 mg·L-1       (nominal concentrations)                 (95% confidence limit: 72.6 – 132.2 mg·L-1)

72 hour – NOECr ˂ 5 mg·L-1            (nominal concentrations)

Key value for chemical safety assessment

EC50 for freshwater algae:
98.6 mg/L
EC10 or NOEC for freshwater algae:
5 mg/L

Additional information

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