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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:
28.08.-10.09.2009
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:
2008
Deviations:
yes
Remarks:
The study was performed without the analytical determination of the test substance concentrations. For all evaluations and results nominal concentrations of the test substance were used.
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Strain: Brinkmann 1953/SAG 86.81
- Source (laboratory, culture collection): from the collection of autotrophic organisms of The Botanic Institute of the Czech Academy of Science, Třeboň, on date 6.5.2008.
- Age of inoculum(at test initiation): 3 days
- 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. The strain culture was always set to pre-culturing of cells for 3 days before the start of the test. Inoculum culture was kept 3 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: none
- Culturing media and conditions (same as test or not): none
- Any deformed or abnormal cells observed: not observed
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
23 ± 2 °C
pH:
Preliminary test: 7.8
Limit test: 7.9
Conductivity:
Preliminary test: 0.116 mS·m-1
Limit test: 0.107 mS·m-1
Nominal and measured concentrations:
Preliminary test: 100, 50, 10, 5, 1 mg/l
Limit test: 100, 91, 83, 75, 68, 62 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks with stoppers venting air
- Type: open
- Material, size, headspace, fill volume: glass
- Aeration: without
- Initial cells density: 10000 cells per ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
The water with conductivity smaller than 5 μS·cm-1 was used for the preparation of solutions. All chemicals used were of analytical-grade.
According to guidancefour basic stock solutions were prepared. The solutions were kept in the dark at temperature of 4±1 °C in fridge. The temperature was checked every day.
The test medium was prepared by admixing 10 mL of basic stock solution A and 1 mL of basic stock solutions B, C, D in 1000 mL deionized water.

OTHER TEST CONDITIONS
Volume of tested mixture: 50 mL
- Light intensity and quality: continuous, min. 6 000 lux, max. 10 000 lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Burker 's counting chamber

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: 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) of the test substance in test medium was prepared for preliminary and limit test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
other: EbC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
other: ErC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: NOECb
Effect conc.:
91 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
other: NOECr
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Results with reference substance (positive control):
- Results with reference substance valid? yes
The sensitivity of the test species and correctness of test performance is periodically verified on a three-month period by testing the reference substance, potassium dichromate. The results of the verification test with K2Cr2O7, carried out in period from 22.6. to 25.6.2009, are the following:

72 hour – ErC50 = 0.74 mg/L (95 % confidence limit: 0.71 – 0.77 mg/L)
72 hour – EbC50 = 0.37 mg/L (95 % confidence limit: 0.35 – 0.39 mg/L)

The given results for the ErC50 are convenient to range of the results of the interlaboratory tests. The result of the last interlaboratory test arranged by Centre for Assessment of Laboratories of T.G. Masaryk Water Research Institute Prague in 2008 for potassium dichromate for 72h – ErC50 was:

72 hour – ErC50 = 0.50 – 1.18 mg/L

The evaluation of the last interlaboratory test arranged by Centre for Assessment of Laboratories of T.G. Masaryk Water Research Institute Prague in 2008 for potassium dichromate was performed only according growth rate. Therefore we give the results for EbC50 of the last interlaboratory test, which are stated in Council Regulation (EC) No. 440/2008, published in O.J.L 142, 2008, method C.3 - Algal Inhibition Test. The result for 72h – EbC50 was :

72h – EbC50 = 0.20 – 0.75 mg/L

The inhibition of test organisms in the reference substance solution meets criteria determined by the interlaboratory tests.
Validity criteria fulfilled:
yes
Conclusions:
The highest inhibition of cell growth in the limit test was 34.5 %. The highest inhibition of growth rate in the limit test was 8.9 %. Therefore exact values of EbC50 and ErC50 could not be calculated and the values of EC are given in the form of a range.
The determination of NOEC values was done by ANOVA (Analysis of Variance) analysis. Used ANOVA method is the part of statistical software QC.Expert 2.5 © 1998-2000 (TriloByte, Czech Republic).
The nominal concentrations of the test substance were used for all evaluations and results.

72 hour - EbC50 > 100 mg/L (nominal concentration)
72 hour - ErC50 > 100 mg/L (nominal concentration)

72 hour - NOECb = 91 mg/L (nominal concentration)
72 hour - NOECr = 100 mg/L (nominal concentration)
Executive summary:

The test substance, Semi Dry Absorption (SDA) Product, was tested for growth inhibition on algae.

The test was performed according to method C.3 - Algal Inhibition Test, Council Regulation (EC) No. 440/2008, published in O.J.L 142, 2008.

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 test medium was prepared for preliminary and limit test.

The preliminary test was performed in range of test substance nominal concentrations 1 – 100 mg·L-1. The highest inhibition of cell growth was 14.7 % in the preliminary test, therefore the limit test was performed in appropriate concentration range. The limit test was performed in range of test substance nominal concentrations 62 – 100 mg·L-1.

The study was performed without the analytical determination of the test substance concentrations. The nominal concentrations of the test substance were used for all evaluations and results. Only the concentrations of metals in stock saturated solution in the limit test were determined.

Test results:

72 hour - EbC50 > 100 mg·L-1        (nominal concentration)

72 hour - ErC50 > 100 mg·L-1         (nominal concentration)

72 hour - NOECb = 91 mg·L-1          (nominal concentration)

72 hour - NOECr = 100 mg·L-1       (nominal concentration)

Description of key information

The test was performed according to method C.3 - Algal Inhibition Test, Council Regulation (EC) No. 440/2008, published in O.J.L 142, 2008.

GLP Study.

Key value for chemical safety assessment

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

Additional information

Test results:

72 hour - EbC50 > 100 mg/L (nominal concentration) (biomass base)

72 hour - ErC50 > 100 mg/L (nominal concentration) (growth rate base)

72 hour - NOECb = 91 mg/L (nominal concentration) (biomass base)

72 hour - NOECr = 100 mg/L (nominal concentration) (growth rate base)

Classification of the test substance on the basis of hazard to aquatic environment was performed according to the Directive 93/21/EEC, Annex IV (article 5.2.1.).

According to the classification criteria given in quoted Directive the test substance, Semi Dry Absorption (SDA) Product, is not assigned to any toxicity categories.