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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The initial concentrations are above the limit of solubility of the test material. The growth kinetics of the solvent control is different from the negative control and the lower test concentrations.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
not specified
Analytical monitoring:
yes
Test organisms (species):
other: other algae: Selenastrum capricornutum [now called Pseudokirchneriella subcapitata ]
Details on test organisms:
- Test Organisms:  
a) Supplier/Source: Test Algae were obtained from American Type Culture  Collection.  
b) Method of Cultivation: Test Algae were cultivated in the sterile  culture.  
c) Stain Number: ATCC22662  
d) Any pretreatment: Test Algae were acclimated for 3 days before  testing.
Test type:
static
Water media type:
freshwater
Limit test:
no
Test temperature:
22.2 - 23.9 ºC
pH:
7.9 - 10.5
Details on test conditions:
- Test Conditions:  
a) Medium: OECD medium  
b) Exposure Vessel Type: 100 mL Medium in a 500 mL Erlenmeyer Flask with glass cap  
c) Nominal Concentrations: control, solvent control (HCO-40; 48 mg/L), 0.25, 0.50, 1.00, 1.60, 2.60, 4.00 and 8.00 mg/L   
d) Vehicle/Solvent and Concentrations: HCO-40: All test solutions contained 48 mg/L HCO-40.  
e) Stock Solutions Preparations and Stability: 500 mg/L stock solution was prepared.  
f) Number of Replicates: 3  
g) Initial Cell Number: 10,000 cells/mL  
h) Water Temperature: 23 ±2 ºC  
i) Light Condition: 4,000 - 5,000 lux, continuously
 j) Shaking: 100 rpm- Analytical Procedure: Test concentrations were measured at the start and the 72nd hour by High Performance Liquid Chromatography method.

- Statistical Method:  a) Data Analysis: The EC50 values and associated 95 % confidence limits were determined by least squares linear regression analysis of the logarithm of nominal test concentration against percent growth inhibition relative to the solvent control.  b) Method of Calculating Mean Measured Concentrations (i.e. arithmetic  mean, geometric mean, etc.): Mean measured concentration was not  calculated in the report.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.28 mg/L
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.54 mg/L
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
7.68 mg/L
Basis for effect:
growth rate
Remarks on result:
other: (95 5 CI = 6.06-10.5 mg/L)

- Measured Concentrations: The tested concentrations were
measured at the start and the 72nd hour. Some of the
measured concentrations were < 80 % of the nominal
concentrations.

-----------------------------------------------------------
Nominal   Measured conc.  %  of nominal   Geometric mean 
conc.          mg/l
--------------------------------------------------------- 
mg/L        0 hrs  72 hrs  0 hrs   72 hrs     

Control     <0.05    <0.05    --     --         0
Solv.Contr. <0.05    <0.05    --     --         0
0.25         0.22     0.09    90     35         0.14
0.50         0.42     0.19    85     37         0.28
1.00         0.79     0.37    79     37         0.54
1.60         1.22     0.54    76     34         0.81
2.60         2.20     0.90    85     35         1.41
4.00         3.40     1.42    85     36         2.2
8.00         7.37     3.55    92     44         5.12
---------------------------------------------------------


Toxicity values estimated by logit method based on the mean measured concentration

- Effect Data:
Area Method
EbC50(0-72h) = 2.21 mg/L (1.98 - 2.52 mg/L)
NOEC = 0.81 mg/L

Rate Method
ErC50(0-72h) = 7.68 mg/l (6.06-10.5 mg/L)
ErC50(0-48h) = 5.03 mg/L (4.41-5.92 mg/L)
ErC50(0-24h) = 9.45 mg/L (6.91-14.6 mg/L)
ErC50(24-72h)= 6.43 mg/L (5.41-8.04 mg/L)
NOEC (0-72hr)= 0.28 mg/L 

The lowest EC50 values observed during the test were 2.21 mg/l (for  biomass) and during the 24-48h interval: an ErC50 of 3.97 mg/l (growth  rate).


- Percent Growth Inhibition of Selenastrum capricornutum

-----------------------------------------------------------
Nominal           Area under the growth curves (Average)
Concentration      Area           Inhibition (%)*1
mg/L              A (0-72hr)      IA (0-72hr)
-----------------------------------------------------------

Control           16,768,000        --
Solvent Control   19,439,000        --
0.25              17,660,000       9.2
0.50              17,444,000       10.3
1.00              17,362,000       10.7
1.60              16,500,000       15.1*
2.60              13,928,000       28.4**
4.00              10,400,000       46.5**
8.00               2,740,000       85.9**
-----------------------------------------------------------


-----------------------------------------------------------                Growth rates and percent inhibition (Average)
Nominal      -----------------------------------------------
Concentration  Rate    Inhibition(%)*1  Rate Inhibition(%)*1 mg/L         u(24-48hr)  Im(24-48hr)  u(24-72hr)  Im(24-72hr) 
-----------------------------------------------------------

Control          0.0748      --          0.0526     --
Solvent Control  0.0722      --          0.0493     --
0.25             0.0730      -1.1        0.0483     2.0
0.50             0.0749      -3.7        0.0483     2.0
1.00             0.0737      -5.0        0.0474     3.9*
1.60             0.0731      -2.9        0.0464     5.9*
2.60             0.0698       3.3*       0.0474     3.9*
4.00             0.0577      20.1*       0.0465     5.7**
8.00             0.0249      65.5*       0.0241     51.1**
-----------------------------------------------------------

*1: Values are the percent inhibition relative to the
solvent control. 
*: Indicates a significant difference (alfa = 0.05) from the
solvent control.
**: Indicates a significant difference (alfa = 0.01) from
the solvent control.


- Growth Curves: Log phase during the test period

Conclusions:
In a valid guideline study, the EbC50(0-72h) was = 2.21 mg/L and the ErC50(0-72h) was = 7.68 mg/l  to Pseudokirchneriella subcapitata.
Executive summary:

In a valid guideline study, the EbC50(0-72h) was = 2.21 mg/L and the ErC50(0-72h) was = 7.68 mg/l  to Pseudokirchneriella subcapitata.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-04-26 - 2010-08-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study OECD201, GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
(2006)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 4.25 - 6.01 - 9.20 - 12.0 - 16.9 µg/L
- Sampling method: 10 mL of each (diluted) sample were added with 100μL acetonitrile and approx. 1 g sodium chloride in a 20 mL headspace vial
prior to analysis. Only the samples at test start were diluted, the samples at test end were analysed directly.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until start of analysis, if necessary.
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 2 - 5 * 10E+3 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.
- Culturing media and conditions (same as test): Nutrient medium Z according to LÜTTGE et al. (1994).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
Nominally 21 - 24 °C, controlled at ±2 °C
pH:
7.90 - 7.92
Nominal and measured concentrations:
Saturated solution with 4 dilution levels: 100 - 50 - 25 - 12.5 - 6.25 %
Initial measured concentrations: 284 - 144 - 84.7 - 36.1 - 18.1 µg/l
Geometric mean measured concentrations during the test period (0 - 72h): 16.9 - 12.0 - 9.20 - 6.01 - 4.25 µg/l
Details on test conditions:

TEST SYSTEM
- Test vessel: 500 ml Erlenmeyer flasks with dilution water (OECD 201 medium)
- Initial cells density: approx. 2 -5 x 10³ cells/ml
- Control end cells density:
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes (OECD 201 medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guidelines

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: pH was stable in the range of 7.67 to 7.71 (highest test concentration) and 7.90 to 7.92 (controles), respectively
-Room Temperature: 21.5 ± 0.5 °C
- Photoperiod: 24 h/d light
- Light intensity and quality: 60 - 120 µE*m(E-2)*s(E-1)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable):

DETERMINATION OF THE TEST ITEM
The concentrations of the test item were analytically determined from all test concentrations and the control after 0 h (test start) and 72 h (test end). Separate samples were prepared at test start without algae. At end of the test samples were taken directly from the test replicates.

CHRONOLOGICAL TEST DESCRIPTION
- Preparation of the saturated solution (experimental starting)
- Application
- Incubation
- Determination of cell density after 0, 24, 48 and 72 h
- Determination of the test item after 0 and 72 h
- Evaluation
CALCULATIONS
All calculations were based on the fluorescence values of each replicate.
Cell density was calculated out of fluorescence values. The fluorescence values were related to cell density values according to a calibration curve.



TEST CONCENTRATIONS

Measured Exposure Concentrations

The saturated solution and further 4 dilution levels of the test item Isodecyl methacrylate and
the control were analytically verified via GC-FID of samples at test start (fresh media, 0 h) and
at test end (old media, 72 h). All effect values are given based on the geometric mean measured concentrations of the test item Isodecyl methacrylate, determined to be: 4.25 – 6.01 – 9.20 – 12.0 – 16.9 μg/L. At test end, all concentrations were below LOQ.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate p.a.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
12 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
16.9 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 16.9 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
9.2 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
12 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
14.1 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
The study meets the validity criteria of the guideline:
- The cell growth increased 184-fold after 72 h in the control (required: at least 16-fold).
- The temperature during the test was in the range of 21 – 24 °C.
The difference from the mean was within ± 2 °C: min.: 21 °C, max.: 22 °C
mean value: 21.5 °C.
- The pH-value in the control replicates increased not higher than 1.5 units: from 7.90 to
7.92.
- The mean coefficients of variation for section-by-section specific growth rates (days
0-1, 1-2 and 2-3) in the control cultures was 6.34 % (required: < 35 %).
- Coefficient of variation of average specific growth rates during the whole test period in
replicate control cultures was 2.53 % (required: < 7 %).
Results with reference substance (positive control):
- Results with reference substance valid?: Yes
- EC50 (0-72 h, biomass growth inhibition): 0.33 (0.318 - 0.350 - [95% confidence interval]), nominal
- EC50 (0-72 h, rate related inhibition): 0.61 (0.551 - 0.679 [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 March 15 to 18, 2010.
Reported statistics and error estimates:
STATISTICS

NOEC/LOEC was determined by calculation of statistical significance of growth rates and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s Test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first.
The SHAPIRO-WILK-Test was used to test for normally distributed populations. P-values for both Normality and Equal Variance test were 0.05. The α-value
(acceptable probability of incorrectly concluding that there is a difference) is α=0.05. Detection of statistically significant differences was considered
negligible when inhibition values were ≤ 10 %. These findings were marked “biologically not significant”. Following the recommendations of the UBA
(HEGER ET AL. 1998), inhibitions > 10 % are significant.

EC-values and statistical analyses: EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals EC10-, EC20- and EC50-values were carried from the best fit values, the standard error and the t-distribution with the software Graph Pad Prism.

NOEC, LOEC and EC values of Isodecyl methacrylate (0 – 72 h) based on geometric mean measured test item concentrations [µg/L]

 

 

 

Rate-related inhibition

 

NOEC

12.0

LOEC

16.9

ErC10

13.2 (12.2 – 15.3)

ErC20

> 16.9

ErC50

> 16.9

 

 

Inhibition of yield

 

NOEC

9.2

LOEC

12.0

EyC10

10.1 (8.33 – 11.3)

EyC20

11.2 (9.75 – 12.5)

EyC50

14.1 (12.6 – 16.1)

 

Validity criteria fulfilled:
yes
Conclusions:
In this study Isodecyl methacrylate was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h.
The NOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were 12.0 and 9.20 μg/L, respectively. The LOEC-values for
inhibition of specific growth rate and inhibition of yield after 72 h were 16.9 and 12.0 μg/L, respectively. The EC50-values for inhibition of specific
growth rate (ErC50) and yield (EyC50) after 72 h were > 16.9 and 14.1 (12.6 – 16.1) μg/L, respectively. All effect levels are given based on geometric mean measured concentrations of Isodecyl methacrylate.
Executive summary:

The toxicity of Isodecyl methacrylate to the unicellular freshwater green algaDesmodesmus subspicatuswas determined in a GLP study according to the principles of OECD 201. The aim was to assess the effects on growth rate and yield over a period of 72 h.The study was conducted under static conditions with an initial cell density of approximately 2 – 5 x 103 cells/mL. Based on a preliminary test, a definitive test with the saturated solution and further four dilution levels were tested in a geometrical series with a dilution factor of 2, nominal: 6.25 - 12.5 - 25 - 50 - 100 % of the saturated solution corresponding to geometric mean measured test item concentrations of 4.25 – 6.01 – 9.20 – 12.0 – 16.9 μg/L. Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of Isodecylmethacrylate were analysed at all dilution levels after 0 and 72 h via Solid Phase Micro Extraction Gas Chromatograhy with FID detection. Initial measured concentrations were 18.1 – 36.1 – 84.7 – 144 and 284 μg/L. After 72 h all measured concentrations were below the limit of quantification (LOQ). All effect values are given based on geometric mean measured concentrations of the test item calculated with half of the LOQ for test end.

Results Synopsis:

Test organism: Desmodesmus subspicatus

Test type: static

72 -hr EC50 (growth rate inhibition, biomass growth inhibition): > 16.9 µg/L (geometric mean measured concentration)

72 -hr NOEC (growth rate inhibition, biomass growth inhibition): 12.0 µg/L (geometric mean measured concentration)

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.

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
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. QA statement)
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.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP, analytical monitoring, closed vessels
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
At the start of the test, samples were taken of each test solution, using the excess remaining after filling the test vessels, and were analyzed for test substance concentration. At the end of the test each blank solution was sampled and analyzed in the same manner.
Vehicle:
no
Details on test solutions:
Two stock solutions were prepared by direct addition of the test substance to 1000 ml of sterile culture medium to give final concentrations of 410 and 10000 mg/L. The three lowest concentrations were prepared by addition of aliquots of the nominal 410 mg/L solution to sterile medium, to a final volume of 1000 ml. The remaining test concentrations were prepared by addition of aliquots of the nominal 10000 mg/l solution to sterile medium, to a final volume of 1000 ml. Test solutions were all clear and colorless.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test organisms were from laboratory cultures maintained under axenic conditions. A culture in the exponential growth phase was used as inoculum
for the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
standard test medium used
Test temperature:
24°C
pH:
start of test 6.39-8.21
end of test 6.11 - 9.69
Nominal and measured concentrations:
Nominal = 0.86, 1.9, 4.1, 9.1, 20, 45 and 100 mg/L
Geometric mean measured = 0.85, 2.0, 3.7, 8.2, 19, 45, 100 mg/L
Details on test conditions:
Test vessels were borosilicate glass bottles of 50 ml nominal capacity, filled completely with airtight, teflon faced disc/crimp closures. Cultures were incubated at 24+/-2C under continous "cool white" illumination with orbital shaking. Four replicates of the control and duplicates of the test substance were employed. At 24, 48 and 72 hours replicate vessels were sacrificed and counted.
Ph was measured at the start of the test and at the end of the test the pH of two repliacte test solutions (containing algae) from each control and test concentration was determined.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
8.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
45 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
20 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
Mean measured concentration of the test substance ranged from 88 -105% of nominal exposure concentrations
Conclusions:
In a valid guideline study, the 72 hr EbC50 and ErC50 were 20 and 45 mg/L, respectively for MAA with Selenastrum capricornutum (new name: Pseudokirchnerella subcapitata). The NOEC for growth and biomass was 8.2 mg/L.
Executive summary:

In a valid guideline study, the 72 hr EbC50 and ErC50 were 20 and 45 mg/L, respectively for MAA with Selenastrum capricornutum (new name: Pseudokirchnerella subcapitata). The NOEC for growth and biomass was 8.2 mg/L.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Study period:
2017-06-20
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
1. SOFTWARE
OECD Toolbox 3.4.0.17
Guideline:
other: REACH Guidance on QSARs R.6
Principles of method if other than guideline:
- Software tool(s) used including version: QSAR-prediction with QSAR Toolbox 3.4.0.17 Database version: 3.8.8/3.1.2
GLP compliance:
no
Specific details on test material used for the study:
SMILES: CCCCCCCCOC(=O)C(C)=C
Test organisms (species):
other:
Details on test organisms:
Species:
Pseudokirchnerella subcapitata (reported as Selenastrum capricornutum)
Selenastrum capricornutum (new name: Pseudokirchnerella subcapitata)
other algae: Selenastrum capricornutum [now called Pseudokirchneriella subcapitata ]
Pseudokirchnerella subcapitata
Desmodesmus subspicatus
Test type:
other: QSAR predicted value
Total exposure duration:
72 h
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.171 mg/L
Nominal / measured:
not specified
Conc. based on:
other: QSAR predicted value
Basis for effect:
growth rate
Remarks on result:
other: QSAR predicted value

QSAR TOOLBOX 3.4.0.17 (database version: 3.8.8/3.1.2) predicted that n-octyl methacrylate has a NOEC of 0.171 mg/L (Algae: Pseudokirchneriella subcapitata; Desmodesmus subspicatus).

Toxicity of the target chemical is predicted from category members using trend analysis based

on 14 values within the range 0.0120 - 400 mg/L from 14 category members. Category members are single

chemicals or mixtures and are selected based on the profile of the target chemical. Only chemicals having

experimental data are listed in the category.

The target chemical FALLS within applicability domain of the prediction.

The data used for calculating the current prediction is taken from 22 experimental values selected from

the following database(s):

1. ECHA CHEM

Validity criteria fulfilled:
yes
Conclusions:
NOEC (72h) algae for n-octyl methacrylate predicted by QSAR TOOLBOX 3.4.0.17 database version: 3.8.8/3.1.2 from category trend analysis of 14 category members is 0.171 mg/L.
Executive summary:

QSAR Toolbox prediction based on trend analysis

Prediction of NOEC Algae (72h) for n-octyl methacrylate

Summary

QSAR TOOLBOX 3.4.0.17 (database version: 3.8.8/3.1.2) predicted that n-octyl methacrylate has a NOEC of 0.171 mg/L (Algae: Pseudokirchneriella subcapitata; Desmodesmus subspicatus).

Toxicity of the target chemical (NOEC = 0.171 mg/L) is predicted from category members using trend analysis based

on 14 values within the range 0.0120 - 400 mg/L from 14 category members. Category members are single

chemicals or mixtures and are selected based on the profile of the target chemical. Only chemicals having

experimental data are listed in the category.

The target chemical FALLS within applicability domain of the prediction.

The data used for calculating the current prediction is taken from 22 experimental values selected from

the following database(s):

1. ECHA CHEM

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.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
(Q)SAR
Adequacy of study:
key study
Study period:
2017-06-20
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
1. SOFTWARE
OECD Toolbox 3.4.0.17
Guideline:
other: REACH Guidance on QSARs R.6
Principles of method if other than guideline:
- Software tool(s) used including version: QSAR-prediction with QSAR Toolbox 3.4.0.17 Database version: 3.8.8/3.1.2
GLP compliance:
no
Specific details on test material used for the study:
SMILES: CCCCCCCCOC(=O)C(C)=C
Test organisms (species):
other:
Details on test organisms:
Species:
Pseudokirchnerella subcapitata (reported as Selenastrum capricornutum)
other algae: Selenastrum capricornutum [now called Pseudokirchneriella subcapitata ]
Pseudokirchnerella subcapitata
Selenastrum capricornutum (new name: Pseudokirchnerella subcapitata)
Desmodesmus subspicatus
Scenedesmus subspicatus (new name: Desmodesmus subspicatus)
Test type:
other: QSAR predicted value
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.05 mg/L
Nominal / measured:
not specified
Conc. based on:
other: QSAR predicted value
Basis for effect:
growth rate
Remarks on result:
other: QSAR predicted value

QSAR TOOLBOX 3.4.0.17 (database version: 3.8.8/3.1.2) predicted that n-octyl methacrylate has an EC50 = 1.05 mg/L( Daphnia magna).

Toxicity of the target chemical is predicted from category members using trend analysis based

on 12 values within the range 0.0169 - 110 mg/L from 12 category members. Category members are single

chemicals or mixtures and are selected based on the profile of the target chemical. Only chemicals having

experimental data are listed in the category.

The target chemical FALLS within applicability domain of the prediction (see Section 4.3 for details).

The data used for calculating the current prediction is taken from 19 experimental values selected from

the following database(s):

1. ECHA CHEM

Validity criteria fulfilled:
yes
Conclusions:
EC50 (48h) Daphnia magna of n-octyl methacrylate predicted by QSAR TOOLBOX 3.4.0.17 database version: 3.8.8/3.1.2 from category trend analysis of 12 category members is 1.05 mg/l.

The predicted EC50 of 1.05 mg/L for n-octyl methacrylate is slightly above the limit for classification.
Isodecyl methacrylate and 2-ethylhexyl methacrylate are at the upper boundary of the log Kow axis close to the target chemical n-octyl methacrylete. Isodecyl methacrylate, having a higher log Kow value between 6.5 and 7.5, has an EC50 around 0.02 mg/L, but the value is less exact due to the low solubility of the substance. 2-ethylhexyl methacrylate is structural closely related to the target chemical and has a similar log Kow. The EC50 for 2-ethylhexyl methacrylate is in the range between 1 and 10 mg/L and thus underpinning the predicted value for n-octyl methacrylate of 1.05 mg/L. Nevertheless, the prediction model is showing a coefficient of determination above 0.75 and is therefore regarded as reliable.
Executive summary:

QSAR Toolbox prediction based on trend analysis

 

Prediction of EC50 (48h) Daphnia magna for n-octyl methacrylate

 

Summary

 

Toxicity of the target chemical (1.05 mg/L) is predicted from category members using trend analysis based on 12 values within the range 0.0169 - 110 mg/L from 12 category members. Category members are single chemicals or mixtures and are selected based on the profile of the target chemical. Only chemicals having experimental data are listed in the category.

The target chemical FALLS within applicability domain of the prediction.

The data used for calculating the current prediction is taken from 19 experimental values selected from the following database(s):

1.   ECHA CHEM

 

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.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included as attachment. Please also see attached justification.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The constituents of the UVCB substance NUMA (Nonyl-Undecyl methacrylate) are structurally related mono alkyl methacrylate esters differing only in the respective alcoholic moieties. The main proportion of these esters are of the n-type the minor proportion of the iso type. Considering the small amount of iso-types and the negligible differences in (eco-) toxicological properties between the n- and iso types, in this assessment both types of one ester with one specific chain length are assessed together as a whole.
Further information is included as attachment. Please also see attached justification Chapter 1

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Further information is included as attachment. Please also see attached justification Chapter 1.

3. ANALOGUE APPROACH JUSTIFICATION
Please see attached justification Chapter 1 and 3.

4. DATA MATRIX
Please see attached justification Chapter 1.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Key result
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
> 0.017 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.012 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Conclusions:
Regardless of the concentration of the various constituents, toxicity on algae for NUMA is derived by direct read across from the constituent i-DMA in a worst case approach.
Executive summary:

The toxicity for aquatic organisms is expected to be at a maximum in the range of C8, C9, C10 and C11 methacrylates.

This hypothesized “turning point” is clearly visible when comparing toxicity of the 72h-algae-studies. The NOECs decrease from 0.28 mg/L for 2-EHMA to 0.012 mg/L for DMA to > 10 mg/L for LMA indicating an increasing bioavailability triggered by polarity. In contrast, the water solubilty decreases from 3.0 mg/L to 0.006 mg/L. Thus for LMA the NOEC is already above the water solubility of the substance. In the case of DMA the solubility is in the same concentration range like the LOEC. In the case of 2-EHMA the water solubility is one order of magnitude higher than the NOEC.

Therefore, regardless of the concentration of the various constituents, toxicity on algae is derived by direct read across from the constituent i-DMA in a worst case approach, leading to a NOEC of 12µg/L and aLOEC of >17µg/L for NUMA.

Description of key information

The toxicity for aquatic organisms is expected to be at a maximum in the range of C8, C9, C10 and C11 methacrylates.

This hypothesized “turning point” is clearly visible when comparing toxicity of the 72h-algae-studies. The NOECs decrease from 0.28 mg/L for 2-EHMA to 0.012 mg/L for DMA to > 10 mg/L for LMA indicating an increasing bioavailability triggered by polarity. In contrast, the water solubilty decreases from 3.0 mg/L to 0.006 mg/L. Thus for LMA the NOEC is already above the water solubility of the substance. In the case of DMA the solubility is in the same concentration range like the LOEC. In the case of 2-EHMA the water solubility is one order of magnitude higher than the NOEC.

Therefore, regardless of the concentration of the various constituents, toxicity on algae is derived by direct read across from the constituent i-DMA in a worst case approach, leading to a NOEC of 12µg/L and a LOEC of >17µg/L for NUMA.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.012 mg/L

Additional information