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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:
2011-2012
Reliability:
2 (reliable with restrictions)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: white solid
Details on test material:
Batch 2237 JG; Expiry date: June 2012.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The stability of tested concentrations in WAF samples was checked in fresh (T=0) and old media (T=72 h).

Test solutions

Vehicle:
no
Details on test solutions:
A special solubilisation protocol was carried out before the experimentation according to guidance document OECD 23 on aquatic toxicity testing of difficult substances and mixtures. This method has consisted in stirring three test item nominal concentrations (1, 10 and 100 mg/L) in test dilution water during 24 hours, at obscurity and 20+/-2°C (each concentration was prepared by adding directly the test item in a flask containing test solution). Each test solution plus a dilution water control have been filtered through a membrane filter to remove suspended particles; solutions obtained were named WAF (Water Accommodated Fraction).

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Freshwater unicellular green algae Pseudokirchneriella subcapitata of the chlorococcal order (formerly named Raphidocelis subcapitata) strain number CCAP 278.4

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
A first algae test had been realised but chemical analysis was not performed on time because of a technical problem. A second Algae test was performed to obtain chemical analysis.

Test conditions

Test temperature:
Test 1: 21.1-23.0°C
Test 2: 22.1-22.6°C
pH:
Test 1: 7.8-8.4
Test 2: 7.6-8.2
Nominal and measured concentrations:
Concentrations measured only for the second test:
Nominal test item concentration: 1 mg/L test item.
Measured at T0: < 0.08 mg/L APG (below LOQ)
Measured at T72h - biotic samples: 0.89 mg/L APG.
Measured at T72h - abiotic samples: < 0.08 mg/L APG (below LOQ)
Measured at T72h - abiotic samples kept in cooler and in the dark: < 0.08 mg/L APG (below LOQ)

Nominal test item concentration: 10 mg/L test item
Measured at T0: 1.57 mg/L APG.
Measured at T72h - biotic samples: 0.91 mg/L APG (-42%).
Measured at T72h - abiotic samples: 0.34 mg/L APG (-78%)
Measured at T72h - abiotic samples kept in cooler and in the dark: 1.56 mg/L APG (-1.0%)

Nominal test item concentration: 100 mg/L test item
Measured at T0: 12.61 mg/L APG.
Measured at T72h - biotic samples: 4.87 mg/L APG (-61%)
Measured at T72h - abiotic samples: 4.64 mg/L APG (-63%)
Measured at T72h - abiotic samples kept in cooler and in the dark: 12.80 mg/L APG (+2%)
Details on test conditions:
The test contained a total of 21 erlenmeyers as follows:
- unfiltered control: 6 replicates
- filtered control: 6 replicates
- Concentration 1 mg/L: 3 replicates
- Concentration 10 mg/L: 3 replicates
- Concentration 100 mg/L: 3 replicates

Inoculum pre-culture:
4 days prior to the test, a pre-culture of algae was run in the same conditions than the test, in order to have a log phase algae culture for inoculation. Before inoculation, this pre-culture was checked under a microscope with a haemocytometer in order to control the algae morphology and to measure the cell number.

Incubation:
A growing exponential phase inoculum of the freshwater unicellular alga Pseudokirchneriella Subcapitata have been added in each erlenmeyer in order to have an initial concentration of 10000 algae/mL at the start of the test: 100 mL of algae cultures have been grown in 250 mL erlenmeyer flasks which have been placed in a cooling incubator at a temperature in the range of 21 to 25°C controlled at +/-2°C and under a constant shaking fixed at 120 rpm and a continuous illumination comprised between 4440 and 8880 Lux.
The algae growth inhibition in relation to the control has been evaluated over a period of 72 hours. The algae concentrations have been measured during the test at 24, 48 and 72 hours with a particle counter.
pH of each test solutions has been measured at the start and at the end of the test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Test N°1:
Inhibition of specific growth rate (mean of three replicates):
-1.1% at 1 mg/L of test item
-4.5% at 10 mg/L of test item
22.1% at 100 mg/L of test item

Test N°2:
Inhibition of specific growth rate (mean of three replicates):
-0.6% at 1 mg/L of test item
-2.1% at 10 mg/L of test item
17.3% at 100 mg/L of test item
Results with reference substance (positive control):
The last algae test performed on the reference item K2Cr2O7 gave an ErC50 (0-72h) = 1.20 mg/L (1.10-1.50 mg/L), indicating a correct sensitivity of the algae which conforms well within the accepted interval of 1.19 mg/L +/- 0.27 fixed by International Standard ISO 6341 May 1996 as mentioned in the OECD Guideline and in accordance with the historical results of the Laboratory.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Static test with WAF approach.
For test item 2237 JG, on Pseudokirchneriella subcapitata (72h):
ErC50 > 100 mg/L (nominal)
NOEC = 10.0 mg/L (nominal)
LOEC = 100.0 mg/L
Executive summary:

In a static acute toxicity test performed according to OECD guideline 201, the alga Pseudokirchneriella subcapitata was exposed for 72 hours to three nominal concentrations ranging between 1 and 100 mg/L of 2237 JG/L. The initial cell concentration was approximately 10 000 cells/mL.

Since the test item was insoluble in the tested medium, the use of Water Accommodated Fractions (WAF) for the preparation of the test media was necessary.

The test item stability was checked by the determination of Alkyl Poly Glucosid (APG) in fresh and old WAF (GC-FID; non GLP data). Because of the instability of APG under light conditions, actual concentrations of APG were not in good agreement with the initial concentrations (-42% and -61% after 72 hours by comparison to initial measurements for 10 and 100 mg/L test concentrations respectively).

The results obtained showed that at the highest tested concentration of 100 mg/L (nominal concentration), a toxicity of the test item with an inhibition of the average specific growth rate in comparison to the control equal to 22.1% during the first test and 17.3% during the second test.

 

ErC50(72 h) > 100 mg/L (nominal)

NOEC (72 h) > 10 mg/L (nominal)