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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:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
Adopted March 23, 2006; Annex 5 corrected 28 July 2011
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium diisopropylnaphthalenesulphonate
EC Number:
215-343-3
EC Name:
Sodium diisopropylnaphthalenesulphonate
Cas Number:
1322-93-6
Molecular formula:
C16H20O3S.Na
IUPAC Name:
sodium diisopropylnaphthalene-sulfonate
Test material form:
solid: particulate/powder
Details on test material:
white to off-white lumps

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Sampling method:
Frequency at t=0 h and t=72 h
Volume 3.5 mL
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at 32 mg/L but without algae and samples for analysis were taken at the start and at the end of the test period.
Additionally, reserve samples of 3.5 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:
no
Details on test solutions:
The batch of CH04207 tested consisted of white to off-white lumps with a purity of >97% and completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with the highest concentration of 100 mg/L. No other treatment than vigorous shaking was needed to completely dissolve 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.
Any residual volumes were discarded.

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella 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
- 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
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 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Test temperature:
between 21 and 23°C
pH:
8.0 - 8.2
Nominal and measured concentrations:
The measured concentrations at the start of the test were 9.6, 18, 32, 57 and 100 mg/L in 10, 18, 32, 56 and 100 mg/L, respectively. During the exposure period, the concentrations remained stable, i.e. were at the level of 98-104% relative to initial at the end of the test.
Based on these results, effect parameters were expressed as analytically confirmed nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Static
- aeration: continuous
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 281.9 x 10^4 cells/mL
- No. of organisms per vessel: 1 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 1 x 10^4 cells/mL
- No. of vessels per control (replicates): 1 x 10^4 cells/mL
- No. of vessels per vehicle control (replicates): 1 x 10^4 cells/mL

GROWTH MEDIUM
- Standard medium used: yes
M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:

TEST MEDIUM / WATER PARAMETERS
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous (24h)
- Light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 87 to 91 µE.m-2.s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
pH At the beginning and at the end of the test.
Temperature of medium Continuously in a temperature control vessel.
Appearance of the cells At the end of the final test, microscopic observations were performed on the highest test concentration to observe for any abnormal appearance of the algae.
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.


TEST CONCENTRATIONS
- Range finding study (0.10, 1.0, 10 and 100 mg/L)
- Test concentrations: Six replicates of exponentially growing algae were exposed to a control and a concentration of 100 mg/L.
- Results used to determine the conditions for the definitive study: A final test was performed based on the results of a preceding combined limit/range-finding test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
56 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
86 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
14 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
32 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
cell number
Details on results:
Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were 9.6, 18, 32, 57 and 100 mg/L in 10, 18, 32, 56 and 100 mg/L, respectively. During the exposure period, the concentrations remained stable, i.e. were at the level of 98-104% relative to initial at the end of the test.
Based on these results, effect parameters were expressed as analytically confirmed nominal concentrations.
The concentrations measured in the samples taken from the solution with algae were comparable with the concentrations measured in the samples without algae. Hence, it can be stated that the presence of the algae did not affect the concentration of the test item in test medium throughout the test.

Inhibition of growth rates and yield increased with increasing concentration of CH04207, resulting in 12% and 44% inhibition, respectively, at the highest test concentration. Statistically significant inhibition of growth rates and yield was found at test concentrations of 18 mg/L and higher. However, growth rate inhibition was considered to be biologically not relevant at 18, 32 and 56 mg/L, where the observed inhibition was below 10%. The NOEC for inhibition of growth rate based on biological relevance was thus set at 56 mg/L.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 100 mg/L when compared to the control.
Results with reference substance (positive control):
Potassium dichromate inhibited growth rate of this fresh water algae species at nominal concentrations of 0.18 mg/L and higher.
The 72h-EC50 for growth rate inhibition (ERC50) was 1.30 mg/L with a 95% confidence interval ranging from 1.29 to 1.32 mg/L.
The 72h-ERC50 was within the expected range of 0.92 and 1.46 mg/L as specified in ISO International Standard 8692, February 2012, and the historical range of 0.86 and 2.3 mg/L, which is based on reference tests performed at the Test Facility during the last ten years.
In conclusion, the sensitivity of this culture of Raphidocelis subcapitata was in agreement with ISO International Standard 8692, February 2012 and the historical data collected at Charles River Den Bosch.
Reported statistics and error estimates:
See table below

Any other information on results incl. tables

Parameter (mg/L)

NOEC*

NOEC#

EC10

EC20

EC50

Growth rate

Value

10

56

86

>100

>100

lower 95%-cl

 

 

72

 

 

upper 95%-cl

 

 

103

 

 

Yield

Value

10

10

14

32

>100

lower 95%-cl

 

 

9.9

26

 

upper 95%-cl

 

 

20

39

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, under the conditions of the present study with Raphidocelis subcapitata, CH04207 inhibited growth rate and yield of this fresh water algae species significantly at analytically confirmed nominal concentrations of 18 mg/L and higher.
The 72h-EC10 for growth rate inhibition (ERC10) was 86 mg/L with a 95% confidence interval ranging from 72 to 103 mg/L.
The 72h-EC50 for growth rate inhibition (ERC50) was beyond the range tested.
The 72h-EC10 for yield inhibition (EYC10) was 14 mg/L with a 95% confidence interval ranging from 9.9 to 20 mg/L.
The 72h-EC50 for yield inhibition (EYC50) was beyond the range tested.
The 72h-NOEC for growth rate inhibition was 10 mg/L based on statistical significance and 56 mg/L based on biological relevance.
The 72h-NOEC for yield inhibition was 10 mg/L based on both statistical significance and biological relevance.
Executive summary:

The objectiveofthe study was to evaluate CH04207 for its ability to generate toxic effects in Raphidocelis subcapitata during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10, EC20and 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 CH04207 tested consisted of white to off-white lumps with a purity of >97% and was completely soluble in test medium at the concentrations tested.

A final test was performed based on the results of a preceding combined limit/range-finding test.Six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to 10, 18, 32, 56 and 100 mg/L. The initial algal cell density was 104 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Samples taken from all test concentrations and the control were analysed. The measured concentrationsat the start of the testwere 9.6, 18, 32, 57 and 100 mg/L in 10, 18, 32, 56 and 100 mg/L, respectively. During the exposure period, the concentrations remained stable, i.e. were at the level of 98-104% relative to initial at the end of the test. Based on these results, effect parameters were expressed as analytically confirmed nominal concentrations.

Inhibition of growth rates and yield increased with increasing concentration of CH04207, resulting in 12% and 44% inhibition, respectively, at the highest test concentration. Statistically significant inhibition of growth rates and yield was found at test concentrations of 18 mg/L and higher. However, growth rate inhibition was considered to be biologically not relevant at 18, 32 and 56 mg/L, because it was below 10%. The NOEC for growth rate inhibition based on biological relevance was thus set at 56 mg/L.

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

10

56

86

>100

>100

lower 95%-cl

 

 

72

 

 

upper 95%-cl

 

 

103

 

 

Yield

Value

10

10

14

32

>100

lower 95%-cl

 

 

9.9

26

 

upper 95%-cl

 

 

20

39

 

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