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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:
06 October 2017 - 18 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to other study

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:
28 July 2011
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 powder with lumps
- Test item storage: at room temperature protected from light
Specific details on test material used for the study:
pH (1% in water, indicative range): 5.12 – 4.78
Solubility in water: poor
Stability in water: not indicated

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control.
In addition, the filter containing the undissolved residue was kept for possible analysis.

Frequency: at t=0 h, t=24 h and t=72 h for each test concentration and at t=0 h and t=72 h for the control
Volume: 2.0 mL
Storage: Samples were stored in a freezer (≤ 15°C) until analysis.

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 the limit 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.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: Direct application of the test item to the test medium with weighing and preparation of test solutions performed under dimmed light. A correction was made for the purity/composition of the test item (correction factor of 1.064 was used). All further concentrations reported are based on the pure test item.
Test solutions with nominal loading rates at 1.0, 10 and 100 mg/L were individually prepared. A 3-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected after filtration through a 0.45 μm membrane filter (RC55, Whatman) and used as test concentrations.
- Controls: Test medium without test item or other additives.
- Evidence of undissolved material: 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 vessel of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.

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
- 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; 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 according to NPR 6505 (''Nederlandse Praktijk Richtlijn no. 6505'')

ACCLIMATION
- Acclimation period: 3 days before the start of the test, cells from the algal stock culture were inoculated in M2-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.
- Acclimation medium different from test medium: no, M2 (according to OECD 201)

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
no

Test conditions

Hardness:
24 mg CaCO3/L
Test temperature:
22-23°C
pH:
Start: 7.9-8.0
End: 8.0-8.2
Nominal and measured concentrations:
Nominal concentrations: control and WAFs individually prepared at nominal loading rates of 1.0, 10 and 100 mg/L
Measured/estimated concentration: in WAF prepared at nominal loading rate of 100 mg/L - 0.13 μg/L (time weighted average concentration)

Since the test concentration decreased below the limit of quantification after 24 hours of exposure, the exposure concentrations at t=24h and t=72h were estimated by extrapolation from the calibration curve to be 0.068 and 0.046 μg/L, respectively (i.e. 5.7% and 3.5% relative to intial, resp.).
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass, open, fill volume: 50 mL
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 284.4 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 6 for the the limit concentration and 3 for the lower concentrations
- No. of vessels per control (replicates): 6
- Other:
* 1 extra replicate of each test concentration for sampling purposes, with the exception of the control
* 1 or 2 extra replicates of each test concentration without algae

TEST MEDIUM / WATER PARAMETERS
- Standard test medium used: yes, M2 (according to OECD 201)
- Source of dilution water: Milli-RO water
- Culture medium different from test medium: yes, M1
- Intervals of water quality measurement: pH at the beginning and at the end of the test for the control and the limit concentration. Temperature: continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light intensity and quality: continuous illumination using TLD-lamps with a light intensity within the range of 91 to 93 µE/m^2/s

EFFECT PARAMETERS MEASURED: cell densities (to evaluate growth rate)
- 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 and the extra replicates as background for the treated solutions.
- Appearance of the cells: at the end of the final test microscopic observations were performed on the limit concentration to observe for any abnormal appearance of the algae.

COMBINED LIMIT/ RANGE-FINDING STUDY
- Test concentrations: 1.0, 10 and 100 mg/L (pure test item)
- Results used to determine the conditions for the definitive study: since no biological significant inhibition of growth was observed, the results were used for the determination of effect concentrations.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (September 2017)

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.13 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: TWA conc calculated based on estimated concentrations
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.13 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: NOEC based on biological relevant effects; TWA conc calculated based on estimated concentrations
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: no; at the end of the test a normal and healthy appearance of the algal cells exposed to the limit concentration was observed, when compared to the control
- A statistical relevant effect at a WAF prepared at a nominal loading rate of 100 mg/L was found (0.9%). This effect, however, was below 10% and considered not biologically relevant. No biologically relevant effects were observed at any of the test concentrations and therefore the NOEC was set on the highest test concentration.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: yes
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Concentrations tested: 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L
- 72h-ErC50: 1.1 mg/L (95% CI: 1.1-1.1 mg/L)
- Other: the result fell within the historical data range (0.82 - 2.3 mg/L).
Reported statistics and error estimates:
An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate (STUDENT t-test, α=0.05, one-sided, smaller).

No ECx-values could be calculated because the test item proved to be non-toxic (ECx > maximum soluble concentration).

The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Analytical verification of test concentrations:

Samples taken from the WAF prepared at a nominal loading rate of 100 mg/L were analysed. At the start of the test, the measured concentration was 1.2 μg/L. This concentration decreased below the limit of quantification after 24h of exposure. Consequently, exposure concentrations were extrapolated from the calibration curve and estimated to be 0.068 and 0.046 μg/L after 24 and 72h of exposure, respectively (i.e. 5.&% and 3.5% relative to initial). Based on these results, the TWA concentration at the limit test concentration was calculated to be 0.13 μg/L.

Small responses at the retention time of the test item were detected in the chromatograms of the blank QC samples. These responses however were acceptable (maximum contribution to LOQ based on peak area was 11%).

Table 1 Measured concentrations versus nominal concentrations

Time of sampling [hours]

Date of sampling

Date of analysis1

Loading rate2[mg/L]

Concentration analyzed [mg/L]

Relative to initial [%]

0

09 Oct 2017

18 Oct 2017

0

0.000015#

 

 

 

 

100

0.00117

 

 

 

 

100*

0.00135

 

24

10 Oct 2017

18 Oct 2017

100

0.000068#

5.8

 

 

 

100*

0.000076#

5.6

72

12 Oct 2017

18 Oct 2017

0

0.000034#

n.a.

 

 

 

100

0.000046#

4.0

 

 

 

100*

0.000040#

3.0

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

2water accommodated fraction corrected for purity (WAF) prepared at the loading rate;

*Without algae;

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

n.a.     Not applicable

Biological results:

Table 2 Individual growth rates per day

Time

Replicate

ROC-118; WAF (mg/L)

Control

1.0

10

100

0-24 h

1

2.079

n.d.

n.d.

2.102

 

2

2.008

n.d.

n.d.

2.16

 

3

2.118

n.d.

n.d.

2.128

 

4

2.097

 

 

2.131

 

5

2.091

 

 

2.144

 

6

2.134

 

 

2.204

 

 

 

 

 

 

Mean:

 

2.088

n.a.

n.a.

2.145

Std.Dev.:

 

0.0437

n.a.

n.a.

0.0346

n:

 

6

n.a.

n.a.

6

CV:

 

2.1

n.a.

n.a.

1.6

 

 

 

 

 

0-48 h

1

1.956

n.d.

n.d.

1.936

 

2

1.996

n.d.

n.d.

1.945

 

3

1.966

n.d.

n.d.

1.935

 

4

1.965

 

 

1.933

 

5

2.001

 

 

1.975

 

6

1.994

 

 

1.959

 

 

 

 

 

 

Mean:

 

1.98

n.a.

n.a.

1.947

Std.Dev.:

 

0.0195

n.a.

n.a.

0.0167

n:

 

6

n.a.

n.a.

6

CV:

 

1

n.a.

n.a.

0.9

 

 

 

 

 

0-72 h

1

1.872

1.876

1.883

1.86

 

2

1.888

1.872

1.946

1.861

 

3

1.88

1.87

1.919

1.866

 

4

1.873

 

 

1.862

 

5

1.895

 

 

1.872

 

6

1.892

 

 

1.878

 

 

 

 

 

 

Mean:

 

1.883

1.873

1.916

1.866

Std.Dev.:

 

0.0098

0.003

0.0313

0.0071

n:

 

6

3

3

6

CV:

 

0.5

0.2

1.6

0.4

Table 3 Growth rate and percentage inhibition for the total test period

ROC-118

WAFs (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

1.883

0.0098

6

 

1.0

1.873

0.0030

3

0.6

10

1.916

0.0313

3

-1.7

100

1.866

0.0071

6

0.9#

#- effect was statistically significant but biologically not relevant (<10%)

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
See 'Overall Remarks' section.
Conclusions:
No biologically relevant effects were observed at any of the test concentration (e.g. no inhibition >10%).
The 72 -hEC50 for growth rate inhibition was >0.13 μg/L and the 72 -h NOEC for growth rate inhibition was ≥0.13 μg/L, based on the TWA exposure concentration.
Executive summary:

A study was performed to assess the effect of ROC-118 on the growth rate of fresh water algae (Pseudokirchneriella subcapitata) after 72 hours of exposure. The study was conducted in accordance with OECD 201 and GLP. The test item was corrected for purity and test item concentrations were reported as mg pure test item/L.

 

A combined limit/range-finding study was performed with test solutions prepared individually at nominal loading rates of 1.0, 10 and 100 mg/L (corrected for purity) by applying a 3 -day period of magnetic stirring to reach maximum dissolution of the test item in the test medium. The resulting aqueous mixtures were filtered through 0.45 µm membrane filters, whereafter the Water Accommodated Fractions (WAFs) were used for testing.

 

Six replicates of exponentially growing algal cultures with an initial algal cell density of 10^4cells/mL were exposed to a blank control and a WAF prepared at a nominal loading rate of 100 mg/L, and three replicates were exposed to WAFs prepared at nominal loading rates of 1.0 and 10 mg/L, respectively. A replicate without algae was included at the limit concentration. Samples taken from the untreated control and the limit concentration were analysed for confirmation of actual exposure concentrations at the start of the test, after 24 and 72 hours of exposure. Since the test concentration decreased below the limit of quantification after 24 hours of exposure, the exposure concentrations at t=24h and t=72h were estimated by extrapolation from the calibration curve to be 0.068 and 0.046 μg/L, respectively. From these concentrations, a TWA of 0.13 μg/L was calculated for the highest concentration tested.

No biologically relevant effects were observed at any of the test concentration (e.g. no inhibition >10%). The 72 -hEC50 for growth rate inhibition was >0.13 μg/L and the 72 -h NOEC for growth rate inhibition was ≥0.13 μg/L, based on the TWA exposure concentration.

All acceptability criteria were met and the study was considered to be valid.