Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24-Dec-2010 to 04-Jan-2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable

Sampling and analysis

Analytical monitoring:
yes

Test solutions

Vehicle:
no
Details on test solutions:
The test medium of the highest nominal concentration of 100 mg/L was prepared by dissolving 100.71 mg of the test item completely in 1000 mL of test water using intense stirring for 10 minutes at room temperature. The test medium of the highest test concentration was serially diluted with test water to prepare the test media of the lower test concentrations. The test media were prepared just before the start of the test.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test organism used for the study was Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum), Strain No. 61.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany). The algae were cultivated at Harlan Laboratories under standardized conditions according to the test guidelines.

Nygaard et al. recommended describing the taxa within the Genus Raphidocelis HINDAK as:

Raphidocelis subcapitata (KORSIKOV) nov. comb.
Basionym: Ankistrodesmus subcapitatus KORSIKOV
Syn.: Kirchneriella subcapitata KORSIKOV
Syn.: Selenastrum capricornutum PRINTZ
Syn.: NIVA-CHL 1

An inoculum culture was set up three days before the start of the exposure. The algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
not applicable

Test conditions

Hardness:
The water hardness (calculated) of the test water was 0.24 mmol/L (= 24 mg/L as CaCO3).
Test temperature:
The water temperature during the test was maintained at 22 °C.
pH:
At the start of the test, the pH measured in the treatments was between 8.0 and 8.1. At the end of the test, pH values of 7.9 to 8.1 were measured (see attached Table 5).
Dissolved oxygen:
not applicable
Salinity:
according to OECD test guideline
Nominal and measured concentrations:
The nominal concentrations of the test item of 1.0, 3.2, 10, 32 and 100 mg/L were tested in parallel with a control.
Details on test conditions:
Since the test item caused a dark colored solution in the test water, 17 mL of test solution were placed in 100-mL Erlenmeyer flasks achieving a reduced layer thickness of 0.9 cm and enhancing the light supply for the algae.

Each test flask was covered with a glass dish. The test flasks were labeled with the study number and all necessary additional information to ensure unique identification. During exposure, the test solutions were continuously stirred by magnetic stirrers.

The test flasks were incubated in a temperature-controlled water bath at a temperature of 22 °C and illuminated by fluorescent tubes (Philips TLD 36W-1/840), installed above the test flasks. The test flasks were positioned randomly and repositioned daily. The mean measured light intensity at the level of the test solutions was approximately 7400 Lux (range: 6820 to 7900 Lux, measured at nine places in the experimental area). The light intensity over the incubation area (measured at nine places in the experimental area) was within a ±15%-deviation from the average light intensity as recommended by the guideline.
The selection of the test concentrations and of the enlarged spacing factor of 3.2 between them was based on the results of range-finding tests (non GLP).

The following nominal concentrations of the test item were tested: 1.0, 3.2, 10, 32 and 100 mg/L. The highest concentration of 100 mg/L corresponded to 72 mg dry matter/L due to a 28% water content of the test item. Additionally, a control was tested in parallel (test water without test item).
The test design included three replicates per test concentration and six replicates of the control.

The test was started using a nominal algal cell density of 10000 cells/mL. The initial cell density was selected according to the recommendations of the OECD test guideline. The algal cell density in the pre-culture was determined by an electronic particle counter (Coulter Counter, Model ZM).

A static test design was applied. The duration of the test was 72 hours.
Reference substance (positive control):
yes
Remarks:
tested twice a year

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and yield
Remarks on result:
other: 95% CL Not stated
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and yield
Remarks on result:
other: 95% CL Not stated
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
93 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% Confidence Limit 88-99 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
28 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% Confidence Limit 23-31 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
37 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% Confidence Limit 34-40 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
19 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% Confidence Limit 7.6-23 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
23 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% Confidence Limit 20-25 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
15 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% Confidence Limit 4.3-20 mg/L
Results with reference substance (positive control):
For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The result of the latest positive control test performed in October 2010 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 0.99 mg/L (Harlan Laboratories study C97564), range of the 72-hour EC50 for the growth rate from 2000 to 2010: 0.71 to 1.7 mg/L).

The test method and the test species are recommended by the test guidelines.
Reported statistics and error estimates:
The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated by Probit Analysis.
For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by Williams t-test.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The test item had a statistically significant inhibitory effect on the growth of the algae (average growth rate and yield) after the test period of 72 hours at the concentration of 32 mg/L and at all higher test concentrations.