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:
13 May 2013 to 18 March 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted according to current test guidelines and GLP compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Name: Oxypyrionacetate
CAS number: 1742-78-6
Batch number: 14058400
Purity: 99.96%
Expiry date: 30 November 2013
Storage details: Refrigerated (2 to 8ºC)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 1.0, 3.2, 10, 32 and 100 mg/L
At the start of the test (0 hours), duplicate 20 mL samples of freshly prepared test media were taken from the control and the test concentration media preparation flasks for chemical analysis.
At 72 hours, duplicate 20 mL samples were taken for chemical analysis from the pooled expired test media from all test concentrations (with and without algae) and control.

Test solutions

Details on test solutions:
The dilution media used was EC medium.
At day -1, a 100 mg/L stock solution of Oxypyrionacetate (parent compound) was prepared and left at room temperature overnight to ensure that the parent compound had fully degraded down to the degradation product (Oxypyrion). The stock was left for a period equivalent to approximately 5 half-lives of the substance which was considered sufficient to ensure the stock contained only the degradation product.
After a pH adjustment of the 100 mg/L stock, the additional concentrations were prepared by serial dilution. A control treatment was prepared by adding EC medium only.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test organism, Pseudokirchneriella subcapitata (Strain 278/4), was originally obtained from the Culture Collection of Algae and Protozoa (CCAP) and is a representative species of the freshwater aquatic phytoplankton. This species is recommended for testing in accordance with the OECD testing guidelines.
Prior to testing, duplicate starter cultures were prepared and incubated under test conditions to obtain sufficient algal cells in exponential growth and to achieve a starting alga cell density of 1 x 104 cells/mL.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not applicable

Test conditions

Hardness:
Not measured
Test temperature:
22.2 – 22.7oC
pH:
6.95 to 8.98
Dissolved oxygen:
Not measured
Salinity:
Not applicable
Details on test conditions:
The test vessels were sterile autoclaved glass 250 mL Erlenmeyer (conical) flasks, into which 100 mL of the appropriate control or test media was added.
Three test vessels were prepared for each test concentration as well as six control vessels (algal nutrient medium only). Each test and control vessel was inoculated with sufficient Pseudokirchneriella subcapitata cells to achieve a starting alga cell concentration of 1 x 104 cells/mL. An additional inoculated vessel was prepared for the control and each test concentration for initial water quality analysis.
An additional test vessel was prepared for each test concentration and the control which were not inoculated with algal cells. These vessels were used to establish background counts on each sampling occasion. Background counts were subtracted from the cell counting results for each of the inoculated test vessels. The resulting cell counts were then used to calculate the area under the growth curves, yield and the corresponding specific growth rates. These vessels were also used for the 72 hour without algae chemical analysis.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
other: EyC50
Effect conc.:
6.419 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 1.618 to 10.05 mg/L
Duration:
72 h
Dose descriptor:
other: EbC50
Effect conc.:
7.254 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: 1.309 to 10.89 mg/L
Duration:
72 h
Dose descriptor:
other: ErC50
Effect conc.:
12.54 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 10.42 to 13.87 mg/L
Details on results:
Based on geometric mean measured concentrations, the 72-hour EyC50 and the 0-72 hour EbC50 and ErC50 values were calculated to be 6.419, 7.254 and 12.54 mg/L, respectively.
The corresponding NOEC values for yield, biomass and specific growth rate after 72 hours were 1.31 mg/L.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
Not applicable

Any other information on results incl. tables

Parameter

Geometric Mean Measured Concentration (mg/L) of Degradation Product Oxypyrion

24-hour

48-hour

72-hour

Yield

-

-

 

EyC10

-

-

1.787
(NC – 2.641)

EyC20

-

-

2.575
(NC – 4.344)

EyC50

-

-

6.419
(1.618 – 10.05)

NOEC

-

-

1.31

Biomass

 

 

 

EbC10

6.124
(0.6432 – 6.148)

1.905
(NC – 9.399)

1.773
(NC – 3.231)

EbC20

7.349
(4.404 – 7.406)

4.592
(NC – 8.706)

2.76
(NC – 6.624)

EbC50

12.44
(10.12 – 12.68)

9.348
(3.148 – 12.65)

7.254
(1.309 – 10.89)

NOEC

5.078

1.31

1.31

Growth rate

 

 

 

EbC10

6.364
(4.519 – 6.425)

4.122
(0.2779 – 7.428)

4.026
(0.9692 – 6.998)

EbC20

7.923
(6.021 – 8.07)

6.228
(2.493 – 8.045)

6.159
(3.891 – 7.603)

EbC50

14.87
(13.53 – 15.53)

12.43
(9.685 – 15.76)

12.54
(10.42 – 13.87

NOEC

5.078

1.31

1.31

-     Not applicable

NC Not calculable

(x – x) 95% confidence limits

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The effects of Oxypyrionacetate on the growth of the unicellular green alga, Pseudokirchneriella subcapitata, were determined during a 72-hour growth inhibition toxicity test conducted in accordance with OECD Chemicals Testing Guideline No. 201 Alga, Growth Inhibition Test (adopted 23 March 2006) (Annex 5 corrected 28 July 2011).
All validity criteria were met therefore the test was considered valid.
As the measured concentrations were outside the range of 80-120% of nominal exposure concentrations of Oxypyrion which is required to report the test concentrations in terms of nominal concentrations, the test concentrations therefore, were reported as geometric mean measured concentrations.
Based on geometric mean measured concentrations, the 72-hour EyC50 and the 0-72 hour EbC50 and ErC50 values were calculated to be 6.419, 7.254 and 12.54 mg/L, respectively, relating to the degradation product Oxypyrion.
The corresponding NOEC values for yield, biomass and specific growth rate after 72 hours were 1.31 mg/L, relating to the degradation product Oxypyrion.
All validity criteria were met therefore the test was considered valid.
Executive summary:

The objective of the study was to determine the effects of Oxypyrionacetate on the growth of the green alga, Pseudokirchneriella subcapitata, during a 72-hour growth inhibition toxicity test. The test was conducted in accordance with OECD Chemicals Testing Guideline No. 201: Alga, Growth Inhibition Test (adopted 23 March 2006) (Annex 5 corrected 28 July 2011).

The definitive test was conducted as at nominal concentration of 1.0, 3.2, 10, 32 and 100 mg/L.The concentration used was based on the results of a range finding test, which is not fully reported.A control group was also included.

Test vessels (250 mL conical flasks) were prepared containing 100 mL of the appropriate test or control medium. Each test vessel was inoculated with 1 x 104 alga cells/mL and incubated at 21 - 24°C (under 4440-8880 Lux light intensity) for 72 hours with cell counts at 24-hour intervals.

The expiry date of the test substance was 30 November 2013; however, the test substance was re-analysed to confirm that the composition had not altered (purity of 99.8%). The test substance was considered acceptable for use on this study.

Initial method development, conducted under Smithers Viscient study 3200294, indicated that at a room temperature of ~20°C, the test substance readily degraded over a period of approximately 3.5 hours. Therefore, due to the test substance rapid degradation it was decided to analyse the degradation product (Oxypyrion) as opposed to the parent compound (Oxypyrionacetate).

Samples of test media were taken for chemical analysis at 0 hour (new test media) and at 72 hours (old test media). 

Analysis of the 0 hour samples showed concentrations of Oxypyrion to range between 63% and 97% of nominal concentrations.

Analysis of the old media at 72 hours showed concentrations of Oxypyrion to range between 24% and 62% of nominal concentrations.

As the measured concentrations were outside the range of 80-120% of nominal exposure concentrations of Oxypyrion, which is required to report the test concentrations in terms of nominal concentrations, the test concentrations were therefore reported as geometric mean measured concentrations.

The 72-hour yield (EyCx) and the 24, 48 and 72-hour biomass (EbCx) and growth rate (ErCx) toxicity values, with corresponding NOEC values, are presented below. Values are presented in terms of measured concentrations.

 

Parameter

Geometric Mean Measured Concentrations (mg/L)
of Degradation Product Oxypyrion

 

24-hour

48-hour

72-hour

Yield

 

 

 

EyC10

-

-

1.787
(NC – 2.641)

EyC20

-

-

2.575
(NC – 4.344)

EyC50

-

-

6.419
(1.618 – 10.05)

NOEC

-

-

1.31

Biomass

 

 

 

EbC10

6.124
(0.6432 – 6.148)

1.905
(NC – 9.399)

1.773
(NC – 3.231)

EbC20

7.349
(4.404 – 7.406)

4.592
(NC – 8.706)

2.76
(NC – 6.624)

EbC50

12.44
(10.12 – 12.68)

9.348
(3.148 – 12.65)

7.254
(1.309 – 10.89)

NOEC

5.078

1.31

1.31

Growth Rate

 

 

 

ErC10

6.364
(4.519 – 6.425)

4.122
(0.2779 – 7.428)

4.026
(0.9692 – 6.998)

ErC20

7.923
(6.021 – 8.07)

6.228
(2.493 – 8.045)

6.159
(3.891 – 7.603)

ErC50

14.87
(13.53 – 15.53)

12.43
(9.685 – 15.76)

12.54
(10.42 – 13.87)

NOEC

5.078

1.31

1.31

-

Not applicable

NC

Not calculable

(x - x)

95% confidence limits

 

Based on geometric mean measured concentrations, the 72-hour EyC50and the0‑72 hour EbC50and ErC50values were calculated to be 6.419, 7.254 and 12.54 mg/L, respectively.

The corresponding NOEC values for yield, biomass and specific growth rate after 72 hours were 1.31 mg/L.

All validity criteria were met therefore the test was considered valid.