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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:
2011-01-17 to 2011-01-20
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:
Commission Regulation (EC) No 761/2009
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
adopted 23rd March, 2006
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: At the start of the experiment three parallel samples were taken from each concentration level and from the control. At the end one sample was taken from each replicate test vessel and one sample was taken from three control container. Representative samples (in duplicate series) were taken at the start and at the end of the experiment.
- Sample storage conditions before analysis: One series was stored frozen (~ -20°C) in the testing laboratory, one series was sent for analysis and was analysed immediately after sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item stock solution was prepared as following: 25.59 mg test item was weighed and suspended in 250 mL dilution water (Mineral Salts Test Medium), thereafter stirred for 2 hours using a magnetic stirrer. After the 2-hour stirring the resulted “stock solution” was centrifuged at 4000 rpm for 10 min. (20 °C) and filtrated using a 1.2 μm filter (Whatman® Glass Microfiber Filter) and further diluted accordingly.
- Controls: yes (untreated control: Algal mineral salts test medium; Toxic reference item: For the evaluation of the quality of the algae and the experimental conditions potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Source: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Nikolausberger Weg 18, D-37073 Göttingen, GERMANY
- Method of cultivation: The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardised conditions according to the test guidelines.


Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
21.4 - 21.9 °C
pH:
pH 7.84 to 7.91 at test start; pH 7.97 to 9.34 at test end
Nominal and measured concentrations:
- geometric mean of the measured concentratins: 0.605, 0.303, 0.144, 0.068, 0.033, 0.018 mg/L
- nominal concentration: TISS/32, TISS/64, TISS/128, TISS/256, TISS/512, TISS/1024 mg/L (TISS; Test item stock solution)
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks of 250 mL volume with 100 mL test medium
- Initial cells density: 10000 algal cells per mL test medium
- Control end cells density: 686700/mL
- No. of organisms per vessel: 100 mL algal suspension per replicate
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The algal mineral salts test medium was prepared according to OECD guideline No. 201
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature: The cultures were maintained at a temperature in the range of 21 – 24 °C controlled at ± 2 °C, which was checked at the beginning of the study and every 24 hours in a flask filled with water. In addition, the temperature was continuously measured (with a min/max thermometer) within the climate chamber.; pH: The pH was checked at the beginning and at the end of the study in the controls and the treated variants.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: yes
- Light intensity and quality: approx. 8000 lux (in average of 8039 lux)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell number was determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscope with a counting chamber.


TEST CONCENTRATIONS
- Range finding study: The concentration levels are chosen based on the results of performed range-finging tests.
- Test concentrations: In the first preliminary range-finding test the nominal concentration range of: 0.78; 1.56; 3.125; 6.25; 12.5; 25; 50 and 100 mg/L concentrations were tested, however instead of the nominal concentrations a more precise description was applied: TISS/128; TISS/64; TISS/32; TISS/16; TISS/8; TISS/4; TISS/2 and TISS mg/L (TISS: Test Item Stock Solution). In the second preliminary range finding test the investigated (nominal) concentrations were: TISS/512; TISS/256; TISS/128; TISS/64 and TISS/32 mg/L..
- Results used to determine the conditions for the definitive study: In the first preliminary range finding test (after 72 hours) an inhibition of 100 % at a concentration of TISS/16 was determined. In the second preliminary range finding test (after 72 hours) an inhibition of 73.3 % at a concentration of TISS/32.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.157 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 0.1363 – 0.1801 (95 % conf. limits)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.439 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 0.3540 – 0.5455 (95 % conf. limits)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.018 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.018 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.033 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.033 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The shape of the algal cells growing was obviously not affected at the control and at the lowest examined concentration of 0.018 mg/L (TISS/1024 mg/L).
In the control the cell density has increased from nominal N= 1 x 10000 cells/mL at the start of the test (0 hours) to N= 68.67 x 10000 cells/mL (mean value) after 72 hours by a factor of 68.67. Thus, the algal growth in the control was high enough to pass the validity criteria in this assay.
Results with reference substance (positive control):
The experimental phase of the last study (Study Number: 392.820.2266) with reference item was: 16-19 August 2010. The report date: 10 September 2010.
The ErC 50 of Potassium dichromate: 1.05 mg/L, (95 % confidence limits: 0.96 – 1.16 mg/L)
The EbC 50 of Potassium dichromate: 0.63 mg/L, (95 % confidence limits: 0.57 – 0.69 mg/L)
The EyC 50 of Potassium dichromate: 0.56 mg/L, (95 % confidence limits: 0.51 – 0.61 mg/L)
The growth rate, 72h value is in accordance with the accepted 1.10 ± 0.48 mg/L range for Pseudokirchneriella subcapitata.
Reported statistics and error estimates:
Mean values and standard deviations were calculated for each treatment at the start, and at the end of the test using Excel for Windows software. Percentage inhibition of area (A), yield (Y) and growth rate (r) were calculated using EXCEL for Windows software. The EC50 values of the test item and their confidence limits were calculated using Probit analysis. The analysis was done using the TOXSTAT statistical software program. For the determination of the LOEC and NOEC, the calculated mean biomass b (area under the growth curve), growth rates r and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test and Williams’test by TOXSTAT software. The data were checked for normality by Chi-Square Test, for homogeneity of variance by Levene’s Test.
Validity criteria fulfilled:
yes
Conclusions:
In the 72-h algal growth inhibition test on Pseudokirchneriella subcapitata with the test item, the 72-h EC50 based on biomass was determined to be 0.1567 mg/L, based on the growth rate 0.4394 mg/L and based on the yield the 72-h EC50 was 0.1252 mg/L. The overall NOEC was determined to be 0.018 mg/L (all calculations were based on the geometric mean of measured concentrations).
Executive summary:

Tert.-Butylperoxy-2-ethylhexanoat was tested in aquatic algae Pseudokirchneriella subcapitata according to EU-Method C.3 (2009) and OECD guideline 201 (2006). At the start of the test the following test item concentrations were measured: TISS/512: 0.053 mg/L; TISS/256: 0.104 mg/L; TISS/128: 0.217 mg/L; TISS/64: 0.418 mg/L; TISS/32: 0.875 mg/L (TISS: Test Item Stock Solution). At the end of the test the measured concentrations were in the range of 41-52 % of the measured start concentrations. The following test item concentrations were measured at the end of the test: TISS/512: 0.021 mg/L; TISS/256: 0.045 mg/L; TISS/128: 0.096 mg/L; TISS/64: 0.219 mg/L; TISS/32: 0.418 mg/L. The TISS/512 and TISS/256 mg/L concentration levels were below the LOQ of the analytical method, however above the LOD and were determined from the extrapolation of the corresponding calibration curve. The lowest examined concentration level of TISS/1024 mg/L was not analytically determined. This concentration level was calculated at the start and at the end of the test, based on the average recovery values and based on the average “measured/start” values. The test item had a statistically significant inhibitory effect on the growth based on the average specific growth rate, yield and areas under the growth curves of Pseudokirchneriella subcapitata after the exposure period of 72 hours in the concentration range of 0.033–0.605 mg test item/L (Bonferroni t-Test (and William’s test), α=0.05). The test concentration of 0.018 mg/L was determined as the 72-hour NOEC. In the 72-h algal growth inhibition test on Pseudokirchneriella subcapitata with tert.-Butylperoxy-2-ethylhexanoat , the 72-h EC50 based on biomass was determined to be 0.1567 mg/L, based on the growth rate 0.4394 mg/L and based on the yield the 72-h EC50 was 0.1252 mg/L. The overall NOEC was determined to be 0.018 mg/L (all calculations were based on the geometric mean of measured concentrations).

Description of key information

Tert.-Butylperoxy-2-ethylhexanoat was assessed in two aquatic toxicity tests to algae according EU method C.3 and OECD Guideline 201. In the key study, tert.-Butylperoxy- 2-ethylhexanoat was tested in aquatic algae Pseudokirchneriella subcapitata according to EU-Method C.3 (2009) and OECD guideline 201 (2006). The test concentration of 0.018 mg/L was determined as the 72-hour NOEC. In the 72-h algal growth inhibition test on Pseudokirchneriella subcapitata with tert.-Butylperoxy-2-ethylhexanoat, the 72-h EC50 based on biomass was determined to be 0.1567 mg/L, based on the growth rate 0.4394 mg/L and based on the yield the 72-h EC50 was 0.1252 mg/L. The overall NOEC was determined to be 0.018 mg/L (all calculations were based on the geometric mean of measured concentrations). In the disregarded study, the concentration range of 0.1862 to 2.4540 mg/L was tested. In this 72-h algal growth inhibition test with Pseudokirchneriella subcapitata (previously known as Raphidocelis subcapitata and Selenastrum capricornutum) , the 72-h EC50 based on growth rate was determined as 0.77 mg/L. The 72-h EC50 based on cell growth was 0.31 mg/L. The overall NOEC was determined to be 0.08 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.44 mg/L
EC10 or NOEC for freshwater algae:
0.018 mg/L

Additional information

In the key study, tert.-Butylperoxy- 2-ethylhexanoat was tested in aquatic algae Pseudokirchneriella subcapitata according to EU-Method C.3 (2009) and OECD guideline 201 (2006). At the start of the test the following test item concentrations were measured: TISS/512: 0.053 mg/L; TISS/256: 0.104 mg/L; TISS/128: 0.217 mg/L; TISS/64: 0.418 mg/L; TISS/32: 0.875 mg/L (TISS: Test Item Stock Solution). At the end of the test the measured concentrations were in the range of 41-52 % of the measured start concentrations. The following test item concentrations were measured at the end of the test: TISS/512: 0.021 mg/L; TISS/256: 0.045 mg/L; TISS/128: 0.096 mg/L; TISS/64: 0.219 mg/L; TISS/32: 0.418 mg/L. The TISS/512 and TISS/256 mg/L concentration levels were below the LOQ of the analytical method, however above the LOD and were determined from the extrapolation of the corresponding calibration curve. The lowest examined concentration level of TISS/1024 mg/L was not analytically determined. This concentration level was calculated at the start and at the end of the test, based on the average recovery values and based on the average “measured/start” values. The test item had a statistically significant inhibitory effect on the growth based on the average specific growth rate, yield and areas under the growth curves of Pseudokirchneriella subcapitata after the exposure period of 72 hours in the concentration range of 0.033–0.605 mg test item/L (Bonferroni t-Test (and William’s test), α=0.05). The test concentration of 0.018 mg/L was determined as the 72-hour NOEC. In the 72-h algal growth inhibition test on Pseudokirchneriella subcapitata with tert.-Butylperoxy- 2-ethylhexanoat the 72-h EC50 based on biomass was determined to be 0.1567 mg/L, based on the growth rate 0.4394 mg/L and based on the yield the 72-h EC50 was 0.1252 mg/L. The overall NOEC was determined to be 0.018 mg/L (all calculations were based on the geometric mean of measured concentrations).

In the disregarded study, 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 tert.-Butylperoxy-2-ethylhexanoat for a duration of 72 hours was performed following the method C3 described in Directive 92/96/EEC of the European Commission and in the Guideline 201 of the OECD.

Algae were exposed to a range of concentrations of tert-butyl 2-ethylperoxyhexanoat 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 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 are as follow:

EbC50 (72h): 0.31 mg/L (0.18 - 0.57)

ErC50 (72h): 0.77 mg/L (0.50 - 1.50)

NOEC: 0.08 mg/L

The disregarded acute study on algal growth inhibition was performed according to OECD guideline 201 (1984) resp. EU method C.3, as well as in compliance with GLP. Nevertheless, due to borderline effects observed and questionable test conditions taken the instability of the test item into account, the validity of this study was called into question. The study data are recorded as disregarded study, the reliability is categorised as Klimisch 4 (not assignable), as a final evaluation and EC50 value deduction is not possible.