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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:
2005-03-01 to 2005-03-04
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Guideline study OECD201, GLP. COMMENTS: In this study dodecyl methacrylate did not cause any inhibiting effects to Desmodesmus subspicatus after 72 h when tested at a concentration of 10 µg/l., which was chosen because it was sufficiently high above the LoQ (1 µg/l) to be measurable, with the consequence that it had to be approx. 10 fold higher than the limit of solubility. Valid with restriction: test concentration above the limit of solubility.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
(1984)
Deviations:
no
Principles of method if other than guideline:
An alga growth inhibition test for determination of the effects of the test item on growth rate and biomass production was carried out over a period of 72 h in a limit test with a nominal concentration of 10 µg/L. This concentration is higher than the solubility in water (< 1 µg/l), but has, nevertheless, been chosen with regard to the feasibility of attaining appropriate and analysable test concentrations at 10 µg/L.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 10 µg/L (limit concentration)
- Sample storage conditions before analysis: Room temperature, protected from light and moisture
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
10.0 mg test item/L was weighed out onto the cap (with PTFE layer) of a 1 L SCHOTT DURAN glass bottle, filled up with 1 L dernineralised water and
shaken at ca. 20 rpm at room temperature for 24 h (rotating shaker 3040, GFL) to get a homogenous emulsion.
- Controls: Six replicates (without test item) were tested under the same test conditions as the test replicates.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus CHODAT SAG 86.81 (formerly known as Scenedesmus subspicatus CHODAT)
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG),
Pflanzenphysiologisches lnstitut der Universität Göttingen,
Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): A four day old preculture was used as inoculum. Incubation was performed in 500 mL Erlenmeyer flasks with
dilution water. For the start of the test the preculture was directly pipetted into the limit concentration and control to receive an initial cell
concentration of approximately 1 * 10E+4 cells/mL in the replicates.
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted 35-70 µE * mE-2 * sE-1 for 24 h per day.


ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not): Nutrient medium Z according to LOTTGE et al. (1994).
- Any deformed or abnormal cells observed:
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
0.48 mmol Ca+Mg/l
Test temperature:
Nominally 23 ±2°C
pH:
8.1 - 8.75
Nominal and measured concentrations:
10.0 µg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Initial cells density:
- Control end cells density:
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guidelines
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio: 3/2
- Conductivity:
- Culture medium different from test medium:
- Intervals of water quality measurement:


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH:
- Photoperiod: 24 h/d light
- Light intensity and quality: 60 - 120 µE * mE-2 * sE-1 (according to 92/69/EC Method C.3)
- Salinity (for marine algae):


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Chlorophyll-impulsfluorometer (Kleinfeld)
- Chlorophyll measurement: Cell density was measured via Chlorophyll-a-fluorescence, excitation at: 435 nm, emission at 685 nm. Each replicate was
measured 6-fold. The cell density was measured at the beginning of the test (after application) and every 24 h. Filtrated culture medium was used as
blank.

- Other: The pH-value at the beginning of the test was measured out of one additional replicate of the limit concentration and control. At the end it
was measured from a pool of all replicates. The room temperature was measured continuously by a hygrotherrnograph. Light intensity was measured prior to test start.
Microscopic evaluation of the cells at the start and at the end of the incubation was performed in order to detect any unusual cell shapes, colour
differences, differences in chloroplast morphology, flocculations, adherence of algae to test containers or aggregation of alga cells.

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: A limit test with a nominal concentration of 10 µg/L was performed. This
concentration is higher than the solubility in water (< 1 ppb), but has, nevertheless, been chosen with regard to the feasibility of attaining appropriate and analysable test concentrations at 10 µg/L.
- Range finding study (NON-GLP)
- Test concentrations: 10 µg/L Results: -1.18 % inhibition of biomass; 0.24% inhibition of growth rate
- Results used to determine the conditions for the definitive study: Based on the results of a preliminary range finding test and the water solubility
of the test item, a definitive test was performed with one limit concentration (10 µg/L) and three replicates.
A control with dilution water (without test item, six replicates) was tested under the same conditions as the test group.
A four day-old preculture incubated at study conditions was used for the main study.
Chlorophyll-fluorescence was determined at the beginning and after 24, 48 and 72 h.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate p.a.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: biomass and growth rate
Remarks on result:
other: 95% confidence interval: not applicable
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 10 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: biomass and growth rate
Remarks on result:
other: 95% confidence interval: not applicable
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 10 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: biomass and growth rate
Remarks on result:
other: 95% confidence interval: not applicable
Results with reference substance (positive control):
- Results with reference substance valid?: Yes
- EC50 (0-72 h, biomass growth inhibition): 0.29 (0.26 - 0.33 (95% confidence interval)), nominal
- EC50 (0-72 h, rate related inhibition): 0.59 (0.55 - 0.68 (95% confidence interval)), nominal
- Other: The toxicity of potassium dichromate to the unicellular freshwater green alga Desmodesmus subspicatus was determined over a period of
72 h from September 14 to 17, 2004.
Reported statistics and error estimates:
EbC50-values and ErC50-values (only test of the reference item) after 72 h were calculated by probit analysis. Probit values according to WEBER
(1986) were used. Calculation of the confidence intervals for EbC50,-values and ErC50-values were carried out using standard procedures according to BREITIG& TÜMPLING (1982).
The NOEC and LOEC were determined by calculation of statistical analyses statistical significance of biomass integrals and growth rates. One Way
Analysis of Variance was carried out for the determination of statistically significant differences compared to control replicates. When running a
One Way Analysis of Variance a Normality Test and an Equal Variance Test were done first. P-values for both Normality and Equal Variance Tests
were 0.05. The a-value for ANOVA was a=0.05.

The recovery rate of the limit concentration was 105% at the start and 94% at 

test end. All effect values are based on the nominal test item concentration.

Validity criteria fulfilled:
yes
Remarks:
Validity criteria: Cell density had to increase at min. 16-fold in the control replicates within 72 h. The temp. during the test had to be in the range of 21-25 °C ±2 °C. pH-value of the control replicates should not deviate by more than 1.5 units.
Conclusions:
In this study the effects of the test item Dodecyl methacrylate on the growth of the freshwater green alga Desmodesmus subspicatus were tested. No
inhibiting effects on biomass growth and specific growth rate were found at the nominal concentration of 10 µg/L. The EC50-values for inhibition of biomass growth (EbC5O) and specific growth rate (ErC50) after 72 h were > 10 µg/L.
Under the conditions of the test Lauryl methacrylate did not inhibit alga growth up to a concentration more than tenfold above the limit of solubility.
Executive summary:

The toxicity of Dodecyl methacrylate (purity: 98.34%) to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD-Guideline 201 (1984) and EC Commission Directive 92169l EC Method C.3 (1992). The aim of the study was to assess the effects on growth rate and biomass production over a period of 72 h. The study was conducted under static conditions with an initial cell density of nominally l0E+4 cells/mL. A limit test with a nominal concentration of 10 µg/L was prepared.

This concentration is higher than the solubility in water (< 1 µg/l), but has, nevertheless, been chosen with regard to the feasibility of attaining appropriate and analysable test concentrations at 10 µg/L. Three replicates were tested for the limit concentration and six replicates for the control. Microscopic evaluation of the cells at start and end of the incubation period revealed no morphological abnormalities. Environmental conditions, pH-value, measured at 0 and 72 h, and room temperature, measured continuously, were determined to be within the acceptable limits. Control analysis was carried out using HPLC at test start and at 24 h intervals. The recovery rate of the limit concentration was 105 % at test start and 94 % at test end. All effect values are based on the nominal test item concentration. Dodecyl methacrylate did not cause any inhibiting effects to Desmodesmus subspicatus after 72 h when tested at a concentration of 10 µg/L.

NOEC and EC50values based on growth inhibition and biomass production were > 10 µg/l, respectively. 

This toxicity study is classified as acceptable and satisfies the guideline requirements for Algae, Growth Inhibition study.

Results Synopsis:

Test organism: Desmodesmus subspicatus

Test type: static

72 -hr EC50 (growth rate inhibition, biomass growth inhibition): > 10 µg/L (95% confidence interval: not applicable)

72 -hr NOEC (growth rate inhibition, biomass growth inhibition): > 10µg/L (95% confidence interval: not applicable)

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Description of key information

There is one study available with acceptable reliability: Dodecyl methacrylate did not cause any inhibiting effects to

Desmodesmus subspicatus after 72 h when tested at a concentration of 10 µg/l.

Key value for chemical safety assessment

Additional information

There is one study available with acceptable reliability: Dodecyl methacrylate did not cause any inhibiting effects to Desmodesmus subspicatus after 72 h when tested at a concentration of 10 µg/l. The test concentration was approx.10-fold higher than the water solubility (technical reasons) setting some limitation to the validity of the study.

The test concentration was chosen because it was sufficiently high above the LoQ (1 µg/l) to be measurable, with the consequence that it had to be approx. 10 fold higher than the limit of solubility.

A further test on dodecyl methacrylate available in the database was considered as invalid because solvent was used and the test concentration was two to three orders of magnitude above the water solubility. In that case, effects by direct contact with undissolved droplets which are not reflecting a concentration-dependency are observed.