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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:
09 - 22 Aug 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2006, corrected 2011
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Württemberg, Karlsruhe, Germany

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction products of trimethylolpropane triglycidyl ether and acrylic acid
EC Number:
603-303-1
Cas Number:
128819-84-1
Molecular formula:
C18 H30 O9, C21 H35 Cl O10, C24 H38 O12 and C27 H43 Cl O13 (mainly)
IUPAC Name:
Reaction products of trimethylolpropane triglycidyl ether and acrylic acid

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: control, 0.763, 2.44, 7.81, 25, and 80 mg/L (nominal)
- Sampling method: Analytical samples were taken at 0 h (initial value) from fresh test solutions and after 72 h (aged solutions). For each sampling also a retain sample was taken. One replicate per treatment with a volume of approximately 500 mL with algae was run in parallel which was used for the analytical sampling.
- Sample storage conditions before analysis: All samples were stored deep frozen at ≤ - 18 °C until they were transferred to the analytical laboratory.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The necessary amount of test item for preparing the stock solution S1 was weighed on a weighing scoop and transferred to a volumetric flask. Test medium was added up to the bench mark and the solution was homogenised by shaking and 30 min of treatment with ultrasonication.
- Differential loading: No, lower test solutions were prepared by dilution of appropriate solutions with test medium.
- Controls: Yes, test medium without test item.
- Evidence of undissolved material: After homogenisation the solution was turbid. Dilutions V1 to V5 (80.0 - 0.763 mg/L) were clear and transparent after preparation.

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Single cell green alga
- Strain: SAG 61.81
- Source: In-house culture, originally purchased from the commercial supplier MBM Sciencebridge GmbH, Göttingen, Germany.
- Age of inoculum (at test initiation): 3 - 4 d before the start of the test, the test medium was inoculated with the test organism and held under test conditions in order to produce a pre-culture in the state of exponential growth.
- Method of cultivation: A semi-continuous liquid stock was held in sterile cultures in the laboratory at 21 - 24 °C and continuous illumination (approximately 4440 - 8880 lux at cell culture level or 60 - 120 µE m^-2 s^-1). CO2 is supplied by shaking on a rotary shaker at approximately 105 rpm. Old medium is periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state. Stock cultures are ordered regularly from the commercial supplier.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Test temperature:
22.1 - 22.9 °C
pH:
7.44 - 8.15
Nominal and measured concentrations:
control, 0.763, 2.44, 7.81, 25.0, and 80.0 mg/L (nominal)
< LOQ, 92.2 - 98.3% TOC of nominal at 0 h and 85.3 - 185% TOC of nominal at 72 h (measured)
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): Closed with aluminium caps.
- size, fill volume: Size: 100 mL; Fill volume: 50 mL
- Initial cell density: 0.5E+04 cells/mL
- Control end cells density: 33.8E+04 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes, AAP-Medium, according to Annex 3 of OECD 201.

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: No, culture medium same as test medium.
- Intervals of water quality measurement: The pH-values were measured at t = 0 and t = 72 h, the temperature was measured at t = 0, 24, 48, and 72 h and the light intensity was determined at four different positions at test start.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes.
- Adjustment of pH: The pH of the test medium was adjusted to 7.5 ± 0.1, if necessary.
- Photoperiod: Continuous light from the side.
- Light intensity and quality: 73.2 - 83.8 µEm^-2 s^-1. The mean light intensity for all positions was 79.7 µEm^-2 s^-1 at test start with measured values deviating in a range of 5.1 to -8.2% which was within ± 15% of variation as specified by OECD 201.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Fluorimeter (fluorescence microplate reader, Infinite 200Pro) at an emission wavelength of 670 nm.
- Other: Morphological apperance (microscope)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: No.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
12.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 11.5 - 14.2
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
7.67 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 6.60 - 8.65 mg/L
Details on results:
- Exponential growth in the control: Yes, cell numbers in the controls were found to increase by a factor of 57.029 between 0 and 72 h, which exceeds the required threshold of 16. This corresponds to a growth rate of 1.35194 d^-1.
- Observation of abnormalities: The cells were considered normal up to and including the nominal test item concentration of 7.81 mg/L. At a concentration of 25.0 mg/L some deformed cells were observed. No cells were observed at 80.0 mg/L nominal test item concentration.
- Any stimulation of growth found in any treatment: Yes, in 0.763 and 2.44 mg/L nominal test item concentrations the inhibition of the growth rate (0 - 72 h) was -8.8 and -9.5%, respectively.
Results with reference substance (positive control):
- Results with reference substance valid? Yes.
- ErC50 (72 h): 1.16 mg/L (nominal, potassium dichromate)
Reported statistics and error estimates:
The statistical evaluation for the 72 h period was performed for growth rate and yield using SAS (2002 - 2010). A test for normality of the data was performed by calculating the Shapiro-Wilk statistic and the homogeneity of variance of the data was evaluated by calcualting the Levene Test. The NoEC and LOEC were determined by using a multiple comparison method (Dunnetts-t-test, left sided, for yield, Bonferroni-Holms corrected Welch test, left sdied, for growth rate). The EC50 -values for growth rate and yield were determined by probit analysis following the normal and logistic distribution, respectively. Due to statiscal reasons inhibition-values above 100% were set to 100 and values below 0% were set to zero. Only concentrations within a clear does response were used for calculations.

Any other information on results incl. tables

ANALYTICAL RESULTS

The measured initial TOC content for the concentrations relevant for the determination of the toxicological endpoints (2.44 mg/L to 80.0 mg/L) ranged from 92.2 - 98.3% of nominal. In the aged samples the measured TOC content for the concentrations relevant for the determination of the toxicological endpoints was between 85.3 - 91.4% of nominal. Since the measured TOC content of concentrations relevant for the determination of the toxicological endpoints were within 80 - 120% of nominal, the toxicological endpoints were evaluated using the nominal concentrations of the test item.

BIOLOGICAL RESULTS

For the inhibition of growth rate and yield a concentration response relation was observed from 2.44 - 80.0 mg/L. The inhibition of growth rate peaked in > 100% at a nominal concentration of 80.0 mg/L.

Table 1. Percentage inhibition of growth rate.

Concentration

[mg/L]

Inhibition of growth rate (0 – 72 h)

[%]

Control

0.0

0.763

-8.8

2.44

-9.5

7.81

10.8 *

25.0

95.4 *

80.0

>100*

* Statistically significantly different to the control

Applicant's summary and conclusion