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 April 2017 - 16 June 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 - 25 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:
22.8 µg/L
Conc. based on:
test mat.
Temp.:
19.9 °C
pH:
>= 8 - <= 8.1
Remarks on result:
other: average of two runs (maximum difference= 27%)
Details on results:
Mean concentration in the water samples (n=10) obtained at the flowrate of 25 mL/h was 25.8 μg/L. CV= 5.9%.
Mean concentration in the water samples (n=10) obtained at the flowrate of 12 mL/h was 19.7 μg/L. CV= 6.0%.

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 19.9°C was determined to be 22.8 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 19.9°C was determined to be 22.8 μg/L. The pH of the aqueous samples was 8.0 -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:
Physical appearance: white crystalline powder
Test item storage: In refrigerator (2-8°C)
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 WAF at 100 mg/L
- Sampling volume: 2 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 concentration before sampling.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test material was a white crystalline powder and not completely soluble in test medium. Test material was added directly to the test medium, at a loading rate of 100 mg/L. A two-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Water Accommodated Fraction (WAF) was collected by filtration through a 0.45 μm membrane filter (RC55, Whatman) and used as test concentrations. 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 104 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): 4 days (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/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
- Culture medium different from test medium: yes, M1
- Pre-culture: 4 days before the start of the limit test, cells from the algal stock culture were inoculated in test medium (M2) at a cell density of 1x10^4 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:
23-24°C
pH:
t=0: 8.1-8.3
t=72: 7.8
Nominal and measured concentrations:
Nominal loading rate: 100 mg/L
Measured concentration: TWA concentration 0.00057 mg/L
The effect parameters are based on the TWA concentration (geometric mean) because the measured concentration decreased during the exposure period from 0.058 mg/L 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: 248 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 WAF at 100 mg/L for sampling after 24 hours of exposure
- 2 replicates of the WAF 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 beginning and end of the test; temperature of the 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 81 to 85 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 cells: at the end of the test, microscopic observations were performed on the WAF 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, WAFs prepared at 1.0, 10, and 100 mg/L.
- 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; reference test 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 abnormalities observed.
- Other:Analysis of the sample taken from the WAF at 100 mg/L at the start of the test showed a measured concentration of 0.058 mg/L. During the exposure period the measured concentration decreased below the limit of detection, i.e. below 0.000041 mg/L, at the end of the test. Based on this result, the TWA concentration was calculated to be 0.00057 mg/L.
- pH and temperature remained within the limits prescribed by the study plan throughout the exposure period.
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:
Two-sample t-test Procedure, α=0.05, one-sided, smaller.
The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

No inhibition of growth rate was observed at the limit concentrations tested, i.e. WAF prepared at a nominal loading rate of 100 mg/L.

Growth Rate And Percentage Inhibition For The Total Test Period

Diona-R, average conc. (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

1.836

0.0365

6

0.00057

1.810

0.0452

6

1.4


     
Growth Rate And Percentage Inhibition At Different Time Intervals

Diona-R

Average conc. (mg/L)

n

0 – 24 h

24 – 48 h

48 – 72h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

6

1.579

0.0

2.033

0.0

1.894

0.0

0.00057

6

1.420

10.1

2.102

-3.4

1.907

-0.7

   
Effect Parameters

Parameter (mg/L)

NOEC

EC10

EC20

EC50

Growth rate

Value

0.00057

>0.00057

>0.00057

>0.00057

Yield

Value

0.00057

>0.00057

>0.00057

>0.00057


Final Test: Test Samples

Time of sampling
[hours]

Date of sampling

Date of
analysis1

Loading rate2

[mg/L]

Concentration
analyzed
[mg/L]

Relative to
initial
[%]

0

06 Jun 2017

16 Jun 2017

0

n.d.

 

 

 

 

100

0.0577

 

 

 

 

 1003

0.0605

 

24

07 Jun 2017

16 Jun 2017

0

n.d.

n.a.

 

 

 

100

0.0000474

0.082

 

 

 

 1003

n.d.

n.a.

72

09 Jun 2017

16 Jun 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     Awater accommodated fraction (WAF)prepared at the loading rate.

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 0.000041 mg/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 of the test substance for inhibition of growth rate (ERC50) of Pseudokirchneriella subcapitata was >0.00057 mg/L, based on TWA concentrations.
The 72h-NOEC for inhibition of growth rate was 0.00057 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 the test substance at nominal loading rate at 100 mg/L (Water Accommodated Fraction) and an untreated control. Exposure concentration in the WAF at 100 mg/L was verified by a validated analytical method to be 0.00057 mg/L (Time Weighted Average). No inhibition of growth rate was recorded at the limit concentration tested. The 72h-ERC50 was >0.00057 mg/L and the 72h-NOEC was 0.00057 mg/L, based on TWA concentrations. The study is considered to be reliable without resitrictions.