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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:
08.02. – 10.03.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Freshwater Algae and Cyanobacteria, Growth Inhibition Test, Commission Regulation (EC) No. 761/2009, Annex IV
Deviations:
yes
Remarks:
(see Any other information ...)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: at the beginning of the test, after 24, 48 hours and at the end of the test in all test concentrations
- Sampling method:
The samples for analysis were prepared at the beginning of the test (0 hours) and immediately delivered in transport box to analytical laboratory.
The samples for analysis after 24 hours and 48 hours were delivered to analytical laboratory immediately after the end of testing period.
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.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Since the test substance is poorly soluble in test medium at concentrations needed for the test, a stock saturated solution (nominal concentration 100 mg·L-1) of the test substance in the test medium was prepared for the preliminary and the limit test.
The stock saturated solution was prepared as follows: the suspension of 100 mg of the test substance in 1000 mL of the test medium was ultrasonicated 30 minutes, stirred for suitable period (72 hours) on a shaft stirrer and subsequently filtered through 0.45 μm filter.
The concentrations of solutions used in the preliminary and the limit test were obtained by dilution of the stock saturated solution with test medium.

- Differential loading:
The results of analytical determinations of concentration of the test substance were in the limit test different from the analytical determination of concentration of the test substance in the preliminary test, therefore the limit test was repeated. For all evaluation and results this limit test No. 2 was used.
The test was performed with nominal concentration 100 mg·L-1. Testing solutions were prepared by dosing of stock saturated solution of the test substance and inoculum into 500 mL volumetric flasks. Testing mixtures were incubated in Erlenmeyers’ flasks.


- Controls: The test contained six parallel series of the test substance and six 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. All chemicals used were of analytical-grade.

- 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: 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 20.5.2015)
- 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.
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.


- Method of cultivation: The strain culture was preinoculated from the stock solution 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.

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:
yes
Total exposure duration:
72 h
Test temperature:
21.5 – 22.5 °C
pH:
7.7
Conductivity:
1.14 µS·cm-1
Nominal and measured concentrations:
100 mg·L-1
Details on test conditions:
Preliminary test
Range of nominal concentration: 1-100 mg/L
Stock solution of the test substance: 100.00 mg/L
Test concentrations: 100, 50, 10, 5 and 1 mg/L
Conductivity of deionized water: 1.84 μS/cm
pH of the test medium: 7.8
Volume of inoculated algae culture: 1.04 mL in 250 mL of mixture
Lighting during the test: 8 320 – 8 360 lux
Temperature during the test: 21.5 – 22.5 °C
Inicial density: 5000 cells per 1mL

Limit test No.2
The full test was performed in a range of the test substance nominal concentrations 9 – 100 mg·L-1. It was observed that the test substance was not completely dissolved and quality criterion 2 was not fulfilled.
Therefore the full test No.2 was performed. The test was performed in range nominal concentrations from 9 mg·L-1 to 100 mg·L-1. For the test was used geometric concentration series of the test substance with factor 1.5. The volume of the test solution was 20 mL in each testing flask.
The full test No.2 conditions:
Stock solution of test substance: 99.97 mg·L-1
Test concentration: 100 mg·L-1
Conductivity of deionized water: 1.14 µS·cm-1
pH of test medium: 7.7
Volume of inoculum algae culture: 6.35 mL in 500 mL mixture
Lighting during the test: 8 320 – 8360 lux
Temperature during the test: 21.5 – 22.5 °C
Inicial density: 5000 cells per 1ml
Testing mixtures were incubated in Erlenmeyers’ flasks. The test contained six parallel series of the test substance concentrations and three controls without the test substance.
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. The growth rates (u) and the yield (Y) and subsequently percentage reduction of growth rate and percentage inhibition of yield were calculated from obtained values.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.145 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 0.145 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
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 = 1.06 mg/L (95% confidence limit: 0.74 – 1.49 mg/L)
Interlaboratory test: 72 hour – ErC50 = 0.51 – 1.19 mg/L

Deviation

There was deviation from the study plan and guideline. The value of EyC50(72h) was not calculated. In the preliminary test the highest inhibition of growth rate was 22.4 % and the highest inhibition of yield was 58.0 %. Based on the results of inhibition of yield the full test should be performed.

Because for the classification and risk assessment of chemicals the value of EyC50(72h) is not needed but only value of ErC50(72h), the limit test was performed based on the results of inhibition of growth rate in the preliminary test.

Validity criteria fulfilled:
yes
Conclusions:
The inhibition of growth rate was 21.4 % in the limit test No.2. Therefore exact value of ErC50 could not be calculated and the value of EC are given in the form of a range.
The determination of NOEC value was done by ANOVA (Analysis of Variance) analysis. Used ANOVA method is the part of statistical software QC.Expert 2.5 © 1998-2000 (product of TriloByte Ltd., Czech Republic).
The guideline specify that if evidence is available to demonstrate that the concentration of the test substance in limit test has been satisfactorily maintained within ± 20 per cent of the nominal or measured initial concentration throughout the limit test, then the results can be based on nominal or measured initial values, which is the case of this study.
The analytical determinations showed, that the concentrations of the test substance were maintained within ± 20% of measured initial values.
The measured initial concentrations were used for all evaluations and results.

Executive summary:

The test substance,2-(3´, 5´-Dichloro-2´-hydroxyphenyl)-4-quinazolinone,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 (EC) No. 761/2009, Annex IV.

 

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 limit 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, stirred for period 72 hours on a shaft stirrer and subsequently filtered through 0.45 μm filter.

 The highest inhibition of growth rate was 22.4 % and the highest inhibition of yield was 58.0 % in the preliminary test.

Based on no toxicity of the test substance found in the preliminary test (measured by growth rate), the limit test was performed subsequently.Only the value of ErC50(72h) is neededforthe classification and risk assessment of chemicals, sothe value of EyC50(72h) was not calculated.

 

In the limit test the results ofanalytical determinationsof concentration of the test substanceweredifferent from theanalyticaldeterminationof concentration of the test substance in the preliminary test, therefore the limit testwas repeated (limit test No.2). For all presentation of results and results the limit test No. 2 was used.

 

The concentration of 100 mg·L-1was tested in the limit test No.2.The inhibition of growth rate was 21.4 %.

 

The guideline specify that if evidence is available to demonstrate that the concentration of the test substance in limit test has been satisfactorily maintained within ± 20 per cent of the nominal or measured initial concentration throughout the limit test, then the results can be based on nominal or measured initial values, which is the case of this study.

The analytical determinationsshowed,thatthe concentrations ofthe test substance weremaintainedwithin ± 20 per cent of measured initial values.

The measured initial concentrations were used for all evaluations and results.

 

Test results:

72 hour – ErC50 > 0.145 mg·L-1       (measured initial concentration)

72 hour – NOECr ˂ 0.145 mg·L-1       (measured initial concentration)

Description of key information

The inhibition of growth rate was 21.4 % in the limit test No.2. Therefore exact value of ErC50 could not be calculated and the value of EC are given in the form of a range.

The determination of NOEC value was done by ANOVA (Analysis of Variance) analysis. Used ANOVA method is the part of statistical software QC.Expert 2.5 © 1998-2000 (product of TriloByte Ltd., Czech Republic).

The guideline specify that if evidence is available to demonstrate that the concentration of the test substance in limit test has been satisfactorily maintained within ± 20 per cent of the nominal or measured initial concentration throughout the limit test, then the results can be based on nominal or measured initial values, which is the case of this study.

The analytical determinations showed, that the concentrations of the test substance were maintained within ± 20% of measured initial values.

The measured initial concentrations were used for all evaluations and results.

Test results:

72 hour – ErC50 > 0.145 mg·L-1      (measured initial concentration)

72 hour –NOECr ˂0.145 mg·L-1      (measured initial concentration)

Key value for chemical safety assessment

EC50 for freshwater algae:
0.145 mg/L
EC10 or NOEC for freshwater algae:
0.145 mg/L

Additional information