Registration Dossier

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:
28 Apr 2017 - 30 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to other study
Reason / purpose:
reference to other study
Reference
Endpoint:
water solubility
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 March 2017 - 26 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Reference:
Composition 0
Qualifier:
according to
Guideline:
EU Method A.6 (Water Solubility)
Version / remarks:
2014
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 105 (Water Solubility)
Version / remarks:
1995
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 830.7840 (Water Solubility)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes
Type of method:
column elution method
Test material information:
Composition 1
Key result
Water solubility:
29.5 µg/L
Conc. based on:
test mat.
Temp.:
20 °C
pH:
>= 7.9 - <= 8.1
Remarks on result:
other: average of two runs (maximum difference=10%)
Details on results:
Mean concentration in the water samples (n=10) obtained at the flowrate of 24 mL/h was 31.0 μg/L. CV= 9.2%.
Mean concentration in the water samples (n=10) obtained at the flowrate of 12 mL/h was 28.0 μg/L. CV= 9.0%.

The coefficient of variation (CV) (n=10) was ≤ 30% at both flow rates. It demonstrated that the system was in equilibrium at all flow rates.

The maximum difference on the mean values at both flow rates was ≤ 30%.

No test substance was detected in the samples from the blank column.

Conclusions:
The water solubility at 20°C was determined to be 29.5 μg/L.
Executive summary:

The water solubility of the substance was determined using the column elution method in a GLP study according to EC A.6, OECD 105 and OPPTS 830.7840.

Quantification was performed by UPLC-MS/MS. The water solubility at 20°C was determined to be 29.5 μg/L. The pH of the aqueous samples was 7.9-8.1.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2009
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Appearance: off-white powder
Test item storage: at room temperature
Specific details on test material used for the study:
Solubility in water: not indicated in the report; please refer to section 4.8 of this dossier
Stability in water: yes

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control and Saturation Solution (SS) at 100 mg/L
- Sampling volume: 2.0 mL
- Sampling frequency: at t=0 h, t=24 h and t=72 h
- Sample storage conditions before analysis: 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 treatment group before sampling.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Direct addition of test substance to test medium at a loading rate of 100 mg/L, applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Saturated Solution (SS) was collected by filtration through a 0.45 μm membrane filter (RC55, Whatman) and used as the highest test concentration. 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 test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.
- Controls: Test medium without test item or other additives.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: in-house laboratory culture.
- Age of inoculum (at test initiation): 3 days before start of the test (pre-culture)

CULTIVATION
- Method: 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.
- Light intensity: 60 to 120 μE/m^2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
- Culture medium different from test medium: yes, M1.
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in test medium (M2) 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.

ACCLIMATION
- Acclimation period: no

Study design

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

Test conditions

Hardness:
0.24 mmol/L (24 mg CaCO3/L)
Test temperature:
22 - 23°C
pH:
8.1
Nominal and measured concentrations:
Nominal loading rate Limit test: 100 mg/L
Measured concentration: TWA (Time Weighted Average) concentration was calculated to be 0.0014 mg/L.
The effect parameters are based on the TWA concentration (geometric mean) because the measured concentration decreased from 0.21 mg/L at test start to
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL test solution, capped.
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 117 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- 1 extra replicate of both the control and the SS at 100 mg/L for sampling after 24 hours of exposure
- 2 replicates of the SS at 100 mg/L without algae.

GROWTH MEDIUM
- Standard medium used: yes, M2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO
- Culture medium different from test medium: yes, M1
- Intervals of water quality measurement: pH: at the beginning and at the end of the test; Temperature of medium: continuously in a temperature control vessel;

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuously
- Light intensity and quality: TLD-lamps with a light intensity within the range of 95 to 106 μE.m^-2.s^-1.

EFFECT PARAMETERS MEASURED:
- Growth rate at 72h
- Determination of cell concentrations: 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 (pathlength =20 mm). Algal medium was used as blank. One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval.
- Other: Appearance of the cells: at the end of the test microscopic observations were performed on the SS at 100 mg/L and the control to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Combined limit-range finding test concentrations: Control, SS at 100 mg/L, 1.0 and 10% of the SS.
- Results used to determine the conditions for the definitive study: yes; the EC50 is expected to exceed the highest loading rate, i.e. 100 mg/L.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (performed Feb 2017)

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.001 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.001 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: No
- Analysis of the sample taken from the SS at 100 mg/L at the start of the test showed a measured concentration of 0.21 mg/L. During the exposure period the measured concentration decreased below the limit of detection, i.e. below 0.00016 mg/L, at the end of the test. Based on this result, the TWA concentration was calculated to be 0.0014 mg/L.
- During the test additional responses related to the test item were observed in the test samples at t=0, t=24 and t=72 hours. The retention times of the additional responses were 0.69, 1.03, 1.16 and 1.20 minutes. At t=0 hours the response observed at the retention time of 1.37 minutes was significant higher than in the QC samples. The additional responses might be degradation products of the test item.
- pH and temperature remained within the limits prescribed by the study plan throughout the exposure period.
- Effect concentrations exceeding solubility of substance in test medium: Yes
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: The EC50 for growth rate inhibition (72h-ERC50) was 1.2 mg/L with a 95% confidence interval ranging from 1.1 to 1.2 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.
Reported statistics and error estimates:
Data for reference substance were generated with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany). Statistical analysis of the test item data was not needed as no inhibition of growth was recorded.

Any other information on results incl. tables

No inhibition of growth rate was observed at the concentration tested, i.e. SS at nominal loading rate of 100 mg/L.

Growth Rate And Percentage Inhibition For The Total Test Period

Norethyl, average conc. (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

1.586

0.0393

6

0.0014

1.623

0.0362

6

-2.3

          
Growth Rate And Percentage Inhibition At Different Time Intervals

Norethyl

Average conc. (mg/L)

n

0 – 24 h

24 – 48 h

48 – 72h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

6

1.622

0.0

1.679

0.0

1.457

0.0

0.0014

6

1.839

-13.4

1.581

5.8

1.447

0.6

Effect Parameters

Parameter (mg/L)

NOEC

EC10

EC20

EC50

Growth rate

Value

0.0014

>0.0014

>0.0014

>0.0014

Yield

Value

0.0014

>0.0014

>0.0014

>0.0014

Final Test: Test Samples

Time of sampling
[hours]

Date of sampling

Date of
analysis1

Percentage of ss2
[%]

Analyzed concentration
[mg/L]

Relative to
initial
[%]

0

15 May 2017

30 May 2017

0

n.d.

 

 

 

 

100

0.214

 

 

 

 

 1003

0.214

 

24

16 May 2017

30 May 2017

0

n.d.

n.a.

 

 

 

100

n.d.

n.a.

 

 

 

 1003

n.d.

n.a.

72

18 May 2017

30 May 2017

0

n.d.

n.a.

 

 

 

100

n.d.

n.a.

 

 

 

 1003

n.d.

n.a.

1     Samples were stored in the freezer (≤ -15°C) until the day of analysis.

2     Percentage of a saturated solution (ss) prepared at a loading rate of 100 mg/L.

3     Without algae.

4     Estimated value, calculated by extrapolation of the calibration curve.

n.d.      Not detected. The limit of detection of the analytical method was determined to be 0.16 µg/L taking a dilution factor of two into account.

n.a.Not applicable.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
For details on validity criteria see 'overall remarks' section.
Conclusions:
The 72h-EC50 for inhibition of growth rate (ERC50) of Pseudokirchneriella subcapitata was >0.0014 mg/L based on TWA concentrations. The 72h-NOEC for inhibition of growth rate was 0.0014 mg/L, based on TWA concentrations.
Executive summary:

In a 72 h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to Norethyl at a nominal loading rate of 100 mg/L (saturated solution) and an untreated control. Exposure concentration was verified by a validated analytical method to be 0.0014 mg/L (TWA). No inhibition of growth rate was recorded at the concentration of Norethyl tested. The 72h-ERC50 was >0.0014 mg/L and the 72h-NOEC was 0.0014 mg/L, based on TWA concentrations. The study is considered to be reliable without restrictions.