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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:
29.01. - 15.02.2018
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 substance concentration was performed at the beginning and at the end of the test. The samples for analysis were taken from the highest, the lowest test concentration and from the highest 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 were analyzed on the day of delivery. 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 Propatyl nitrate 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 facility.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The test substance is poorly soluble in the test medium at concentrations needed for the test. For this reason, the preliminary experiment was performed for finding suitable period for achieving the highest solubility.
For the preliminary experiment the solutions were prepared as follows: 100 mg of the test substance in 1000 mL of test medium was ultrasonicated for 30 minutes and stirred for period 3, 24, 48 and 72 hours on a shaft stirrer, subsequently filtered through 0.45 μm filter and the concentrations of the test substance in resulted solutions were determined.
On the basis of the measured concentrations of the test substance, the period of 72 hours stirring was chosen.

Final procedure for the preparation of the test solutions was then set as follows:
The saturated solutions of the test substance were prepared in the test medium. The saturated solutions were ultrasonicated for 30 minutes and stirred for period 72 hours and subsequently filtered through 0.45 μm filter.
The concentrations of test solutions used in the preliminary and the full test were obtained by dilution of the saturated solution with test medium.

- Differential loading: The test was performed in a range measured concentrations from 12.1 mg·L-1 to 61.36 mg·L-1. For the test was used geometric concentration series of the test substance with factor 1.3.

- 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 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. 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. After this period the culture was in the state of the exponential growth and had the cell density suitable for the performance of the test. The cell density of the pre-culture was measured just before the start of the test and the needed inoculum volume was calculated.

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:
2.49 µS·cm-1
Nominal and measured concentrations:
1000, 770, 590, 450, 350, 270 and 210 mL·L-1
210: measured 12.13 mg.L-1 (0 h), 13.09 mg.L-1 (72 h)
1000: measured 59.88 mg.L-1 (0 h), 59.70 mg.L-1 (72 h)
1000 w.a.: measured 61.36 mg.L-1 (0 h), 61.17 mg.L-1 (72 h) without algae
Details on test conditions:
Preliminary test
Preparation of the saturated solution (02.02.2018)
Saturated solutions of the test substance: 49.99 mg / 500 mL-1
Conductivity of deionized water: 2.91 µS·cm-1
pH of the test medium: 8.1
Preparation of the test concentrations (05.02.2018)
The test concentrations: 1000, 500, 100, 50 and 10 mL·L-1
Conductivity of deionized water: 1.97 µS·cm-1
pH of the test medium: 8.0
Volume of inoculum algae culture: 1.82 mL in 200 mL mixture
Lighting during the test: 7015 – 7025 lux
Temperature during the test: 23.0 – 23.5 °C

Full test
Preparation of the saturated solution (09.02.2018)
Saturated solutions of the test substance: 100.02 mg·L-1
Conductivity of deionized water: 1.97 µS·cm-1
pH of the test medium: 8.0
Preparation of the test concentrations (12.02.2018)
The test concentrations: 1000, 770, 590, 450, 350, 270 and 210 mL·L-1
Conductivity of deionized water: 2.49 µS·cm-1
pH of the test medium: 8.1
Volume of inoculum algae culture: 4.21 mL in 200 mL mixture
Lighting during the test: 7020 – 7030 lux
Temperature during the test: 23.0 – 23.5 °C

The test contained 3 parallel series of the test item concentration and 3 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.:
56.4 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 12.1 mg/L
Nominal / measured:
meas. (initial)
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 initial measured concentrations were used for all evaluations and results.
72 hour – ErC50 = 56.4 mg·L-1 (95% confidence limit: 37.2 – 83.9 mg·L-1)
72 hour – NOECr ˂ 12.1 mg·L-1
Executive summary:

The test substance, Propatyl nitrate, 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.

Since the test substance is poorly soluble in test medium at concentrations needed for the test, the saturated solutions of the test substance in test medium were prepared for preliminary and full test. The saturated solutions were prepared by adding measured amounts of the test substance to the test medium. The saturated solutions were ultrasonicated for 30 minutes and stirred for period 72 hours on a shaft stirrer and subsequently filtered through 0.45 μm filter.

The preliminary test was performed in a range of the test substance measured concentrations

0.6 – 55.25 mg·L-1. Based on toxicity of the test substance found in preliminary test, the full test was performed in a range of the test substance measured concentrations 12.1 – 61.36 mg·L-1.

The analytical results showed, that the test substance Propatyl nitrate was sufficiently stable in the test medium at conditions of the test. Samples for analytical determination were taken at the beginning and at the end of the test.

The guideline specify, that if evidence is available to demonstrate that the concentration of the test substance 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 initial measured concentrations were used for all evaluations and results.

 

Test results:

72 hour – ErC50 = 56.4 mg·L-1       (95% confidence limit: 37.2 – 83.9 mg·L-1)

72 hour – EyC50 = 17.7 mg·L-1       (95% confidence limit: 17.6 – 17.8 mg·L-1)

 

72 hour – ErC10 = 10.7 mg·L-1       (95% confidence limit: 7.7 – 14.8 mg·L-1)

72 hour – EyC10 = 7.3 mg·L-1         (95% confidence limit: 7.3 – 7.4 mg·L-1)

 

72 hour – NOECr ˂ 12.1 mg·L-1      

72 hour – NOECy ˂ 12.1 mg·L-1      

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 initial measured concentrations were used for all evaluations and results.

72 hour – ErC50 = 56.4 mg·L-1       (95% confidence limit: 37.2 – 83.9 mg·L-1)

72 hour – NOECr ˂ 12.1 mg·L-1      

Key value for chemical safety assessment

EC50 for freshwater algae:
56.4 mg/L
EC10 or NOEC for freshwater algae:
12.1 mg/L

Additional information