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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-02-06-2001-02-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Remarks:
Not specified in report
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable
Analytical monitoring:
yes
Details on sampling:
MONITORING OF TEST SUBSTANCE CONCENTRATION: after 0, 24, 48 and 72 hours of exposure
Vehicle:
no
Details on test solutions:
Epichlorohydrin was tested at nominal test concentrations of 0, 1.0, 2.2, 5.0, 11, 25 and 100 mg/l in algal medium. Algae were exposed for 72 hours using Erlenmeyer flasks containing 100 ml test solution.

To study the exposure of the algae to epichlorohydrin, samples of the test solutions were taken at the start, day 1, day 2 and on day 3 of the test and analysed with Gas Chromatography. The difference between the nominal and the mean measured concentrations was larger than 20 % and for this reason, the biological endpoints of the test are based on the average of the measured concentration being 0, 0.87, 1.7, 3.9, 8.4, 19 and 40 mg/l.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Strain: ATCC 22662
- Source/Supplier: Culture Collection of Algae and Protozoa,
Ambleside, Cumbria, UK
- Laboratory culture: yes
- Method of cultivation: algae were incubated at 23°C with
continuous illumination and weekly transferred into fresh
medium
- Controls: yes (six replicates)
- Initial cell concentration: 1x10E+04
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Study was terminated after 72 hours
Hardness:
no data
Test temperature:
22.3-23.4°C
pH:
8.0 at start and 8.4 at end of the test
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
Nominal Concentrations: 0, 1.0, 2.2, 5.0, 11, 25 and 55 mg/l
Measure Concentrations: 0, 0.87, 1.7, 3.9, 8.4, 19 and 40 mg/l
Details on test conditions:
GROWTH/TEST MEDIUM CHEMISTRY
- Medium used: according to OECD 201
TEST SYSTEM
- Test type: static without renewal of test solution
- Concentrations: 0, 1.0, 2.2, 5.0, 11, 25 and 55 mg/l
- Exposure vessel type: 100 ml medium in a 250 ml Erlenmeyer
flask with a cap that allowed ventilation
- Number of replicates: three/concentration
- Test temperature: 22.3-23.4°C
- pH: 8.0 at start and 8.4 at end of the test
- Intensity of irradiation: about 4400 lx
- Photoperiod: continuous
- Shaking: about 100 rpm
TEST PARAMETER: inhibition of cell multiplication
MONITORING OF TEST SUBSTANCE CONCENTRATION: after 0, 24, 48 and 72 hours of exposure
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
7.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.7 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
15 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
RESULTS: EXPOSED AND CONTROL
-------------------------------------------------------------
Nominal conc. mean measured conc. mean cell density (day 3)
-------------------------------------------------------------
0 not detected 110 x 10E+4
1.0 0.87 103 x 10E+4
2.2 1.7 110 x 10E+4
5.0 3.9 91.6 x 10E+4
11 8.4 44.8 x 10E+4
25 19 5.86 x 10E+4
55 40 1.90 x 10E+4
-------------------------------------------------------------
- Growth curves: Logarithmic growth until end of the test
(72 h)
Results with reference substance (positive control):
No data
Reported statistics and error estimates:
Not applicable

The cell density increased 81 - 127 times in the controls, which meets the validity criterion of a minimal 16 times increase. The cell density measurements were used to calculate the biomass integral and growth rate for each Erlenmeyer flask. Using linear interpolation, with mean measured concentrations on a logarithmic scale (base = 10), the ECS050 .0-72h for biomass integral and growth rate were 7.1 and 15 mg/l, respectively. The No Observed Effect Concentration (NOEC), based on biomass integral at test termination, was determined with Williams' Test (Williams, 1972). Based on mean measured concentrations this revealed a NOEC of 1.7 mg/l.

Conclusions:
Following a 72 hour exposure, the biomass EC50 is 7.1 mg/L and the NOEC is 1.7 mg/L.
Executive summary:

The toxicity of epichlorohydrin to algae (Selenastrum capricornutum) was tested according to OECD Guideline 201 (OECD, 1984) and according to OECD GLP Principles of Good Laboratory Practice and Compliance Monitoring No. 1, ENV/MC/CHEM/(98)17 of 21 January 1998. Epichlorohydrin was tested at nominal test concentrations of 0, 1 .0, 2.2, 5.0, 11, 25 and 100 mg/l in algal medium. Algae were exposed for 72 hours using Erlenmeyer flasks containing 100 ml test solution.

To study the exposure of the algae to epichlorohydrin, samples of the test solutions were taken at the start, day 1, day 2 and on day 3 of the test and analysed with Gas Chromatography. The difference between the nominal and the mean measured concentrations was larger than 20 % and for this reason the biological endpoints of the test are based on the average of the measured concentration being 0,

0.87, 1.7, 3.9, 8.4, 19 and 40 mg/l.

The cell density increased 81 - 127 times in the controls, which meets the validity criterion of a minimal 16 times increase. The cell density measurements were used to calculate the biomass integral and growth rate for each Erlenmeyer flask. Using linear interpolation, with mean measured concentrations on a logarithmic scale (base = 10), the EC50-72h for biomass integral and growth rate were 7.1 and 15 mg/l, respectively. The No Observed Effect Concentration (NOEC), based on biomass integral at test termination, was determined with Williams' Test (Williams, 1972). Based on mean measured concentrations this revealed a NOEC of 1.7 mg/l.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was conducted in conformance to GLP and in accordance to US EPA guideline, however, there was no analytical confirmation of test solution concentrations in this study.
Qualifier:
according to
Guideline:
other: US EPA 1971. Algal Assay Procedure: Bottle Test. Corvalis, Oregon and US EPA 1978. Selenastrum Capricornutum Printz Algal Assay Bottle Test. EPA - 600/9-78-018. Corvalis, Oregon.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable
Analytical monitoring:
no
Details on sampling:
no data
Vehicle:
no
Details on test solutions:
Test solutions were prepared by appropriate dilution of the stock solution with algal assay medium.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Test type:
static
Water media type:
freshwater
Total exposure duration:
96 h
Hardness:
no data
Test temperature:
24 +/- 2 degrees C
pH:
no data
Dissolved oxygen:
no data
Salinity:
not applicable
Nominal and measured concentrations:
Test concentration range covered an order of magnitude with each concentration in the series being 60& of the next higher concentration.
Details on test conditions:
Test conditions were:
Temperature: 24 C +/- 2 C; light, 400 +/- 80 fc continuous, cool white fluorescent; shaking, 100 rpm; free gas exchange through urethane foam plugs; test volume, 50 ml in 125 ml Erlenmyer flasks. One ml of a 5 x 10^5 cells/ml algal inoculum was added per flask, giving a 1 x 10^4 cell/ml initial cell count.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
16 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: total cell volume
Remarks on result:
other: 12-21
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
17 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: total cell volume
Remarks on result:
other: 13-22
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
13 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 9.8-17
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
16 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 13-20

EC50 values (i.e., the concentration estimated to cause a 50% reduction of a specific growth parameter in a given time interval) were calculated for each test chemical using both cell counts and total cellular volume as effect criteria. Cell count EC5O's were not significantly different from those for TCV.

Conclusions:
Based on total cell volume, the EC50 for epichlorohydrin was 16 mg/L in Test 1 and 17 mg/L in Test 2. Based on cell number, the EC50 for epichlorohydrin was 13 mg/L in Test 1 and 16 mg/L in Test 2.
Executive summary:

The purpose of this study was to evaluate algal test procedures using a standard test system. Seven chemicals were tested in duplicate for their toxicity to the green alga, Selenastrum capricornutum Printz. EC50 values (the concentration estimated to cause a 50% reduction of a specific growth parameter) were determined for these chemicals using both cell counts and total cell volume (TCV) as effect criteria. Duplicate test results indicated good reproducibility of the system. The test chemicals and their 96-hour TCV EC50 values are given below:

EC50 based on total cell volume (95% CONFIDENCE INTERVAL) mg/L

Test 1 16 (12 – 21)

Test 2 17 (13 – 22)

EC50 based on cell number (95% CONFIDENCE INTERVAL) mg/L

Test 1 13 (9.8 - 17)

Test 2 16 (13 – 20)

Description of key information

In a growth inhibition test with Pseudokirchnerella subcapitata, the 72-hour ErC50 for epichlorohydrin was 15 mg/L.  The 72-hour NOEC for epichlorohydrin was determined to be 1.7 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
15 mg/L
EC10 or NOEC for freshwater algae:
1.7 mg/L

Additional information

Two toxicity tests with the green algae, Pseudokirchnerella subcapitata,were assessed for reliability, and both studies were found to be of good quality and reliable for use in the risk assessment process. One algal toxicity study measured both growth rate (r) and biomass (y) as indicators of growth inhibition over 72 hours of exposure.  The 72-hour ErC50 for growth rate in this study with P. subcapitata was 15 mg/L epichlorohydrin. The 72-hour NOEC and EyC50 (based on biomass) in this same study with P. subcapitata were reported as 1.7 and 7.1 mg/L epichlorohydrin, respectively. In the other reliable algal toxicity test with P. subcapitata, the 96-hour EC50 for epichlorohydrin was determined by examining both cell number and total cell volume. Each endpoint was examined in repeat algal tests to examine the precision of the assay. Therefore, the 96-hour EC50 values (based on cell number) were 13 mg/L and 16 mg/L epichlorohydrin, and the 96-hour EC50 values (based on total cell volume) were 16 mg/L and 17 mg/L epichlorohydrin. Since the use of the average specific growth rate for estimating toxicity is scientifically preferred (OECD 201, 2006), the 72-hour NOEC and ErC50 of 1.7 and 15 mg/L epichlorohydrin, respectively, were selected as the key parameters for this endpoint.