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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:
2017-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
Adopted March 23, 2006; Annex 5 corrected 28 July 2011
Deviations:
yes
Remarks:
Cell densities were determined using a spectrophotometer with immersion probe (path length = 10 mm instead of 20 mm). Evaluation: A new spectrophotometer was used. This has no impact on the test interpretation
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
1,1'-[methylenebis(sulphonyl)]diethylene
EC Number:
221-909-0
EC Name:
1,1'-[methylenebis(sulphonyl)]diethylene
Cas Number:
3278-22-6
Molecular formula:
C5H8O4S2
IUPAC Name:
(ethenesulfonyl)methanesulfonylethene
Test material form:
solid: particulate/powder
Details on test material:
white crystalline powder

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency at t=0 h, t=24 h and t=72 h
Volume 2.0 mL
Storage Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
Additionally, reserve samples of 2.0 mL were taken from all test solutions for possible analysis.

Test solutions

Vehicle:
yes
Remarks:
acetonitrile
Details on test solutions:
Preparation of test solutions started with the highest concentration of 10 mg/L (second range-finding test and final test) or 100 mg/L (first range-finding test) applying 11-35 minutes of magnetic stirring to accelerate dissolution of the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

ACCLIMATION
- Culturing media and conditions (same as test or not): same
- Any deformed or abnormal cells observed: no

Study design

Test type:
static
Water media type:
freshwater
Remarks:
M2; according to the OECD 201 Guideline, formulated using Milli-RO water
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
no

Test conditions

Test temperature:
During the exposure period the temperature measured in the incubator was maintained between 21 and 23°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within 2°C).
pH:
7.7-8
Nominal and measured concentrations:
0.010, 0.018, 0.032, 0.056 and 0.10 mg/L.
Samples taken from all concentrations were analysed. At the start of the test, the actual test concentrations were at 38-47% of the nominal concentration if they were within the range of the calibration curve. These concentrations decreased by more than 20% at the end of the test.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Type (delete if not applicable): Capped vessels
- Aeration: no
- Initial cells density: An initial cell density of 1 x 10^4 cells/mL.
- Control end cells density:
- No. of organisms per vessel: 1 x 10^4 cells/mL.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
1 extra replicate of each test group for sampling purposes after 24 hours of exposure,
1 or 2 replicates of each test concentration without algae.


GROWTH MEDIUM
- Standard medium used: yes: M2


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Continuously using TLD-lamps with a light intensity within the range of 94 to 95 µE.m-2.s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: at 680 nm using a spectrophotometer with immersion probe (path length = 20 mm or 10 mm).
- Chlorophyll measurement: no
- Other: At the end of the final test, microscopic observations were performed on the control and nominally 0.056 mg/L to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Range finding study: yes, 2
- Test concentrations:
RF1: 0.1, 1, 10, 100 mg/L
RF2: 0.001, 0.01, 0.1 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.007 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.008 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.022 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.022 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.004 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.004 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
yield
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.011 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.022 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
yield
Details on results:
At the start of the test, the actual test concentrations were at 38-47% of the nominal concentration if they were within the range of the calibration curve. These concentrations decreased by more than 20% at the end of the test. The concentrations measured in the samples without algae were comparable at the start of the test but decreased less during the course of the test, indicating that the presence of biomass has an influence on the test item concentrations. Based on these results, the Time Weighted Average exposure concentrations were calculated and used to determine the effect parameters.

Growth rates were in the range of the controls at the lowest test concentration during the 72-hour test period, whereas the growth rate of algae exposed to the four highest concentrations were increasingly reduced. Statistically significant inhibition of growth rate was found at the four highest test concentrations. The effects observed at TWA concentrations of 0.0040 and 0.0074 mg/L were below 10% and thus considered to be biologically not relevant.
Inhibition of yield increased with increasing concentration of CH02241 resulting in 97% inhibition at the highest test concentration (TWA = 0.028 mg/L). Statistically and biologically significant inhibition of yield was found at TWA test concentrations of 0.0040 mg/L and higher.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 0.012 mg/L when compared to the control.


Results with reference substance (positive control):
The EC50 for growth rate inhibition (72h-ERC50) was 1.1 mg/L with a 95% confidence interval ranging from 1.1 to 1.1 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range.
The EC50 for yield inhibition (72h-EYC50) was 0.36 mg/L with a 95% confidence interval ranging from 0.35 to 0.36 mg/L. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L. Hence, the 72h-EYC50 for the algal culture tested was below this range. Nevertheless, this was accepted as the EC50 for growth rate was within historical range.
Reported statistics and error estimates:
Parameter( mg/l) NOEC(biological rel.) EC10 EC20 EC50
Growth rate Value 0.0074 0.0077 0.011 0.022
lower 95%-cl 0.0072 0.011 0.022
upper 95%-cl 0.0082 0.012 0.023
Yield Value 0.0040 0.0041 0.0053 0.0087
lower 95%-cl 0.0037 0.0049 0.0082
upper 95%-cl 0.0046 0.0058 0.0092

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, under the conditions of the present study with Pseudokirchneriella subcapitata, CH02241 reduced growth rate and inhibited the yield of this fresh water algae species significantly at TWA concentrations of 0.0045 mg/L and higher.
The EC50 for growth rate inhibition (72h-ERC50) was 0.022 mg/L with a 95% confidence interval ranging from 0.022 to 0.023 mg/L.
The EC50 for yield inhibition (72h-EYC50) was 0.0087 mg/L with a 95% confidence interval ranging from 0.0082 to 0.0092 mg/L.
The 72h-NOEC for growth rate inhibition was 0.0040 mg/L based on statistical significance.
The 72h-NOEC for growth rate inhibition was 0.0074 mg/L based on biological relevance.
The 72h-NOEC for yield inhibition was 0.0040 mg/L based on statistical significance.
Executive summary:

The objectiveofthe study was to evaluate CH02241 for its ability to induce toxic effects in Pseudokirchneriella subcapitata during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10and EC50for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011.

The batch of CH02241 tested was a white crystalline powder and completely soluble in test medium at the concentrations tested.

A final test was performed based on the results of two preceding range-finding tests. Six exponentially growing algal cultures were exposed to an untreated control, three replicates per concentration were exposed to 0.010, 0.018, 0.032, 0.056 and 0.10 mg CH02241 per litre. The initial algal cell density was 104cells/mL.The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

A dose-related increase of inhibition of both growth rate and yield was observed at the end of the test. An inhibition of 61% and 97% was observed at the highest concentration tested for growth rate and yield, respectively.

Samples taken from all concentrations were analysed. At the start of the test, the actual test concentrations were0.0030, 0.0072, 0.0149, 0.0248 and 0.0466 mg/L in nominally 0.010, 0.018, 0.032, 0.056 and 0.10 mg/L, respectively. These concentrations decreased to 10-58% of the initial concentrations at the end of the test. Based on these results, the Time Weighted Average exposure concentrations were calculated and used to determine the effect parameters.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters obtained in this study are summarized in the table below.

Parameter (mg/L)

NOEC*

NOEC**

EC10

EC20

EC50

Growth rate

Value

0.0024

0.0062

0.0068

0.010

0.022

lower 95%-cl

 

 

0.0063

0.010

0.021

upper 95%-cl

 

 

0.0073

0.011

0.023

Yield

Value

0.0024

0.0024

0.0036

0.0047

0.0077

lower 95%-cl

 

 

0.0032

0.0043

0.0073

upper 95%-cl

 

 

0.0041

0.0051

0.0082

cl – confidence limit, * based on statistical significance, ** based on biological relevance

 

In conclusion, under the conditions of the present study with Pseudokirchneriella subcapitata, CH02241 reduced growth rate and inhibited the yield of this fresh water algae species significantly at TWA concentrations of 0.0045 mg/L and higher.