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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:
November 23 2005 to February 2 2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Carried out using OECD guideline and under GLP . However, the pH variation of the control was 2.03, which is more than the recommended 1.5, and the variation of the growth rate coefficient in the control solution was 3.5%, which is lower than the recommended 5%.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
the final concentrations of test compound were not maintained within the designated limit of 80 % of the initial concentrations in non-inoculated flasks, variation of growth rate coefficient < 5%
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
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
Vehicle:
no
Details on test solutions:
For the preliminary and definitive tests, a stock solution of the test item in water was prepared 72 h before the beginning of the test (time 0) by mixing 100mg of (2,5-DIMETHYL-2,5-DI(2-ETHYLHEXANOYLPEROXY) HEXANE at 96.4%) in one litre of dilution water at room temperature. In both cases stirring was maintained during the 72 h, then the solution was filtered twice on Millipore HAWP 0.45 micro filter to obtain clear solution.
Ultra-pure water was used for the preparation of dilution water (resistivity> 18MO).
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Unicellular green fresh-water algae Pseudokirchneriella subcapitata, CCAP 278/4 stock (previously named Raphidocelis subcapitata and Selenastrum capricornutum) obtained from the Culture Centre of Algae and Protozoa (Ambleside, UK). Two flasks, each containing approximately 100 ml of axenic stock culture of algae were incubated at 23 ± 1 °C under lighting (photoperiod: 16 hours of illumination, 8 hours of darkness), slowly continuously shaken. These stock cultures were renewed every week, using two new cultures. The quality of the stock culture was verified for the absence of micro-organisms and deformed cells under microscopic observation before use.

3 days before the beginning of the study two pre-cultures were prepared by inoculating each stock suspension of algae (5 ml) into sterile dilution water (500 ml). The pre-cultures were incubated under the same conditions as those used for the stock cultures. Only one of the two pre-cultures was used to inoculate the test flasks; the second one was to be used only if the first one was damaged.
At the beginning of the test, the cell concentration of the pre-culture was determined. The result was used to calculate the volume needed and introduced into each test flask in order to get an initial cell concentration equal to 104 cells/ml.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
not applicable
Hardness:
50 mg/L NaHCO3
Test temperature:
7.90 - 7.99
pH:
8.23 - 10.02
Dissolved oxygen:
8.7 - 9.3
Salinity:
not applicable
Nominal and measured concentrations:
0.1 - 100 mg/L - preliminary test nominal concentrations
100 mg/L - definitive test nominal concentration
Details on test conditions:
Preliminary test

For the preliminary and definitive tests, a stock solution of the test item in water was prepared 72 h before the beginning of the test (time 0) by mixing 100mg of (2,5-DIMETHYL-2,5-DI(2¬ETHYLHEXANOYLPEROXY) HEXANE at 96.4%) in one litre of dilution water at room temperature. In both cases stirring was maintained during the 72 h, then the solution was filtered twice on Millipore HAWP 0.45 micro filter to obtain clear solution.

Flasks were stoppered with cellulose bungs, and placed in the phytoculture cupboard
An inoculated control flask (labelled T) was prepared and incubated under the same conditions, with no test item. This was used for determining algae growth. A non-inoculated blank (labelled 81) containing only dilution water was also prepared and incubated

After 24h, 48h, and 72h of incubation, a volume of 200 IJI was sampled from each test flask, pipetted into a quartz microplaque. Fluorescence was then determined with a cytofluorimeter, and by comparison with a calibration range, cell density was determined, according to the measured fluorescence.

Dissolved O2 and pH were measured in the control and the highest concentration, in non inoculated flask at the beginning of the test and in an inoculated flask at the end of the test.

Results of the preliminary test served to define the concentration range used in the definitive test.

Definitive test
As results of preliminary test did not show any significiant toxicity, a limit test at 100mg/1 (nominal concentration) was prepared.

Inoculated control flasks (labelled T) were prepared and incubated under the same conditions, with no test item to determine algae growth. Non inoculated blanks (labelled BI) containing only dilution water were prepared and incubated. A non inoculated supplementary series of test flasks was also prepared and incubated in order to be chemically analysed at the end of the test period. By comparing the test item concentrations in this series and in the inoculated one, the stability of the test item and its potential absorption, adsorption or metabolization by algae was evaluated.

Test flasks, controls and blanks were prepared in triplicate.

After 24h, 48h, and 72h of incubation, about 1 ml was sampled from each test flask under agitation and introduced into culture tubes. After 24 and 48 hours test flasks were replaced in the same position in the rotary shaker. Culture tubes were stored in darkness until determination of algae concentration by counting as described in § 5.2.1. Measured concentrations of algae were used for calculating the percentages inhibition and plotting the growth curves

pH and dissolved O2 were measured in all concentrations, at the beginning and the end of the test.
Reference substance (positive control):
yes
Remarks:
sodium dichromate (tested every 2 months)
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The appearance of the test solutions was visually checked at the beginning and at the end of the test. Solutions were found tobe clear,colourless, became not cloudy over the period of the test. No precipitation was observed at the end of the test. Microscopic observation confirmed that the algae appeared normal at the end of the test. The normal form of the unicellular algae is a crescent shaped cel lwith an average length of 5-10
Results with reference substance (positive control):
ErC50 = 0.61 mg/L
EbC50 = 0.98 mg/L
According to ISO 8692, the acceptable range is 0.60 - 1.03 mg/L for ErC50 and 0.20 - 0.75 mg/L for EbC50
Reported statistics and error estimates:
The NOEC (No Observed Effect Concentration), is normally determined by statistic multisample analysis using the Dunnett method (US EPA vs 1.5 software). In the case of this study, NOECs could not be determined.

As results of preliminary test did not show any significiant toxicity, a limit test at 100mg/1 (nominal concentration) was prepared.

Validity criteria fulfilled:
yes
Executive summary:

Summary

The determination of the growth inhibition of the freshwater algae Pseudokirchneriella subcapitata(previously known as Raphidocelis subcapitata and Selenastrum capricornutum) exposed to the test item 2,5-Dimethyl-2,5-Di(2-Ethylhexanoylperoxy)Hexane for a duration of 72 hours was performed followingthemethod C.3 described in Directive 92t69tEEC of the European Commission and in the Guideline 201 of the OECD.

 

Algae were exposed to a range of concentrations of 2,5-Dimethyl-2,5-Di(2­ethylhexanoylperoxy)Hexane dissolved in dilution water. The toxic effect measured during the assay was the inhibition of cellular multiplication over a time period of 72 hours.

 

The study was performed using 120 ml glass bottles stoppered with cellulose bungs, containing 50 ml of test solution inoculated with an algal suspension so that the initial cell concentration was equal to 1x104cells/ml. Test flasks were incubated at 23 ± 2 °c continuously shaken and constantly illuminated. The cell density was measured daily. Analytical chemistry and physico­chemical measurements were carried out at the beginning and the end of the test.

 

The concentration of test item causing a 50% reduction in biomass (EbC50 or in growth rate (ErC50) was determined. The No Observed Effect Concentration (NOEC), corresponding to the highest tested concentration where no significant effect was observed in comparison to the control, was determined using a statistical analytical method (Dunnett test). The results were as follows:

 

Effective concentration and NOEC

 

Value

95% C.I.

Cell growth

72 h EbC50

>Solubility limit (0.247 mg/L)

ND

72h EbC10

ND

ND

NOECb

ND

ND

 

 

 

 

Growth rate

 

72 h ErC50

>Solubility limit (0.247 mg/L)

ND

72h ErC10

ND

ND

NOECr

ND

ND

 

 

 

ND – not determined

Description of key information

One OECD 201 study was carried out under GLP is available where ErC50 > 100 mg/L (loading rate). The substance is not toxic up to the limit of solubility.

Key value for chemical safety assessment

Additional information

In the OECD 201 study, algae were exposed to a range of concentrations dissolved in dilution water. The toxic effect measured during the assay was the inhibition of cellular multiplication over a time period of 72 hours. The study was performed using 120 ml glass bottles stoppered with cellulose bungs, containing 50 ml of test solution inoculated with an algal suspension so that the initial cell concentration was equal to 1x104cells/ml. Test flasks were incubated at 23 ± 2 °c continuously shaken and constantly illuminated. The cell density was measured daily. Analytical chemistry and physico­chemical measurements were carried out at the beginning and the end of the test.

Test substance was not toxic up to the limit of solubility