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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 MAY 2010 to 12 NOV 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted in accordance with current testing guidelines and GLP-compliant.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
TOC analysis
Vehicle:
no
Details on test solutions:
The definitive test was conducted at nominal active ingredient concentrations of 1.97, 2.96, 4.44, 6.67 and 10 mg/L (equivalent to 2.63, 3.95, 5.92, 8.89 and 13.3 mg MK92K/L.

On each renewal occasion, a stock medium was prepared at a nominal active ingredient concentration of 100 mg/L by weighing ca 266.66 mg of MK92K and diluting to 2000 mL with ASTM medium. This solution was serially-diluted to prepare a series of stock media at nominal active ingredient concentrations of 66.7, 44.4, 29.6 and 19.7 mg/L. Test media at the concentrations required for the test were prepared by diluting 100 mL of each stock medium to 1000 mL with ASTM (American Society for Testing and Materials - standard hard water).

A control treatment was prepared by adding ASTM medium only to the test vessels.
Test organisms (species):
Daphnia magna
Details on test organisms:
Juvenile Daphnia magna (Straus), (less than 24 hours old), were taken from parental cultures (aged 14 days), held and maintained at Covance Laboratories Ltd. The culture was originally obtained from Institute National de Recherche Chimique Applique (IRChA), France.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
145 - 148 mg/L
Test temperature:
20.7 - 21.3°C
pH:
7.9 - 8.4
Dissolved oxygen:
95 - 102 % ASV
8.32 - 9.31 mg/L
Nominal and measured concentrations:
Nominal active ingredient concentrations of 1.97, 2.96, 4.44, 6.67 and 10 mg/L (equivalent to 2.63, 3.95, 5.92, 8.89 and 13.3 mg MK92K/L). Mean measured active ingredient concentration (calculated based ona MK92K active ingredient : TOC ratio of 10:6:6): 1.62, 2.71, 4.21, 6.70 and 10.8 mg/L.
Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
6.24 other: mg a.i./L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility
Remarks on result:
other: (8.32 mg/L nominal MK92K concentration)
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
3.1 other: mg a.i./L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility
Remarks on result:
other: (4.13 mg/L nominal MK92K concentration)
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
4.44 other: mg a.i./L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility
Remarks on result:
other: (5.92 mg/L nominal MK92K concentration)
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
1.97 other: mg a.i./L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility
Remarks on result:
other: (2.63 mg/L nominal MK92K concentration)

TOC Analysis of MK92K in Test Media
During the definitive test mean measured TOC concentrations of 1.07, 1.79, 2.78, 4.42 and 7.10 ppm were achieved, corresponding to 82.3, 91.8, 94.9, 100 and 108 % of the nominal TOC concentrations. Based on the results of TOC analysis, mean measured concentrations in terms of active ingredient were considered to be equal to 1.62, 2.71, 4.21, 6.70 and 10.8 mg/L. As the measured concentrations were within the 80 to 120% of nominal TOC range, the toxicity of MK92K to Daphnia magna is expressed in terms of the nominal active ingredient concentrations.

Test Media Descriptions

On preparation, stock media at nominal active ingredient concentrations of 19.7, 29.6 and 44.4 mg/L appeared as very pale yellow solutions. At 66.7 mg/L the test medium appeared as a pale yellow solution and at 100 mg/L as a yellow, homogeneous hazy dispersion. An odour was emitted from each stock medium, the strength of which increased with concentration.

The control and test media at all nominal concentrations appeared as colourless solutions for the duration of the test, with the exception of the test media at 10 mg/L that was very pale yellow in colour. At 24 hours, the new test media at concentrations of 4.44 to 10 mg/L emitted an odour. The strength of the odour increased with concentration.

Toxicity to Daphnia magna

Table 1: Immobilisation of Daphnia magna after 24 hours exposure to MK92K during the definitive test

Nominal active ingredient (mg a.i./L)

Number of Daphnia magna exposed

Mobile Daphnia magna

Immobile Daphnia magna

Submerged

Surface

Submerged

Surface

Control

20

19

1

-

-

1.97

20

20

-

-

-

2.96

20

20

-

-

-

4.44

20

19*

-

1

-

6.67

20

5*

-

15

-

10

20

2*

-

18

-

-              Value not appropriate
*
             Daphnia magna observed to be moving more slowly compared to the control

Table 2: Immobilisation of Daphnia magna after 48 hours exposure to MK92K during the definitive test

Nominal active ingredient (mg a.i./L)

Number ofDaphnia magnaexposed

MobileDaphnia magna

ImmobileDaphnia magna

Submerged

Surface

Submerged

Surface

Control

20

19

1

-

-

1.97

20

20

-

-

-

2.96

20

11*

-

8

1

4.44

20

1*

-

19

-

6.67

20

-

-

18

2

10

20

-

-

20

-

-              Value not appropriate
*
             Daphnia magna observed to be moving more slowly compared to the control

No immobility was observed in the control treatment throughout the test.

In terms of nominal active ingredient concentrations of MK92K, the 24 and 48-hour EC50toxicity values for immobility to Daphnia magna are presented below:

 

24-Hour

48-Hour

EC50

6.24 mg a.i./L

(8.32 mg/L)

3.10 mg a.i/L

(4.13 mg/L)

NOEC

4.44 mg a.i./L

(5.92 mg/L)

1.97 mg a.i./L

(2.63 mg/L)

(italics) based on nominal MK92K concentrations

The highest measured concentration at which no significant immobility occurred was 1.97 mg/L.

The lowest concentration at which 100% immobility occurred was 6.67 mg/L.

The validity criteria for control immobility (≤10%) and dissolved oxygen (>60% air saturation value) were both satisfied. The test is therefore considered valid.

Validity criteria fulfilled:
yes
Conclusions:
The 48-hour acute toxicity to the freshwater planktonic crustacean, Daphnia magna, was determined in accordance with the requirements of OECD Chemicals Testing Guideline No. 202 Daphnia sp. Acute Immobilisation Test (adopted April 13 2004).
No immobility was observed in the control treatment.
The highest nominal active ingredient concentration at which no significant immobility occurred was 1.97 mg/L.
The lowest nominal active ingredient concentration at which 100% immobility occurred was 6.67 mg/L.
The validity criteria for control immobility (≤10%) and dissolved oxygen (>60% air saturation value) were both satisfied. The test is therefore considered valid.
Executive summary:

Introduction

The objective of the study was to determine the 48-hour acute toxicity to the freshwater planktonic crustacean, Daphnia magna. The study was conducted at approximately 20°C using a semi-static test regime (renewal of test media at 24 hours).

Methods

A definitive test was conducted at nominal active ingredient concentrations of 1.97, 2.96, 4.44, 6.67 and 10 mg/L (equivalent to 2.63, 3.95, 5.92, 8.89 and 13.3 mg/L), the concentration range was based on the results of a range-finding test.

On each renewal occasion, a stock medium was prepared at a nominal active ingredient concentration of 100 mg/L by weighing ca 266.66 mg and diluting to 2000 mL ASTM medium. This solution was serially-diluted to prepare a series of stock media at nominal active ingredient concentrations of 66.7, 44.4, 29.6 and 19.7 mg/L. Each stock medium was diluted, as appropriate, to give test media at the concentrations required for the test.

At the start of the test, five juvenile Daphnia magna were added to each test vessel. The animals in each test vessel were observed at 24 and 48 hours. The animals were not fed during the test. Samples for total organic carbon (TOC) analysis were taken at 0 and 24 hours from freshly-prepared test media and at 24 and 48 hours from pooled samples of used test media. Daphnia were transferred into fresh test media following observations each day at 24-hour intervals.

Results

All water quality parameters were within the ranges specified in the study guidelines.

Total organic carbon (TOC) analysis conducted during the range-finding test indicated that test solutions prepared at nominal active ingredient concentrations of 10 and 100 mg/L correlated to 6.4 and 68 ppm TOC, thereby indicating active ingredient : TOC ratio of 10:6.6. Following background/control correction it was determined that TOC analysis for MK92K concentrations ≤1.0 mg/L was inaccurate and below the limit of detection for the TOC method.

Based on the results of the range-finding test and a MK92K active ingredient : TOC ratio of 10:6.6 the definitive test was conducted at 1.97, 2.96, 4.44, 6.67 and 10.0 mg a.i./L (equivalent to 1.3, 1.95, 2.93, 4.40 and 6.60 ppm TOC)

Mean measured TOC concentrations of 1.07, 1.79, 2.78, 4.42 and 7.10 ppm were achieved, corresponding to 82.3, 91.8, 94.9, 100 and 108 % of the nominal TOC concentrations. As the measured TOC concentrations were within the 80 to 120% of nominal TOC range, the toxicity of MK92K to Daphnia magna is expressed in terms of the nominal concentrations.

 

The 24 and 48-hour EC50 toxicity values for immobility to Daphnia magna are presented below:

 

24-hour

48-hour

EC50

6.24 mg a.i./L

(8.32 mg/L)

3.10 mg a.i/L

(4.13 mg/L)

NOEC

4.44 mg a.i./L

(5.92 mg/L)

1.97 mg a.i./L

(2.63 mg/L)

(italics)based on nominal concentrations

The highest nominal active ingredient concentration at which no significant immobility occurred following 48 hours exposure was 1.97 mg a.i./L.

The validity criteria for control immobility (≤10%) and dissolved oxygen (>60% air saturation value) were both satisfied. The test is therefore considered valid.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
MAR 2009 to 17 NOV 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Conducted in accordance with current testing guidelines and GLP-compliant, however insufficient test substance details provided.
Qualifier:
according to
Guideline:
other: draft method ISO 14669:1999(E)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
The sample was described as insoluble and so Water Accommodated Fractions, (WAFs) were prepared.

WAFs containing concentrations of 0.63, 1.13, 2.00, 3.56, 6.32 and 11.25 mg/L MK92K were prepared in dilution media (25 µm filtered sea water). After being spun overnight and then left to settle for four hours some sample was found to be floating on the surface of the vessels. 20 mL from the central portion of each was then removed to be used as the test concentration.
Test organisms (species):
other: Acartia tonsa
Details on test organisms:
- Name: Acartia tonsa
- Supplier: Guernsey Sea Farms, Guernsey
- Culture conditions: Test animals were received on 02.09.09 and maintained according to STL Runcorn SOP I.7.
- Test animals: At the start of the test the Acartia tonsa nauplii were 13-15 days old.
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
20 ± 2°C
pH:
7.90
Dissolved oxygen:
100%
Salinity:
33.6 g/L
Nominal and measured concentrations:
0, 2, 20 and 200 mg/L for the range-finder
0, 0.63, 1.13, 2.00, 3.56, 6.32 and 11.25 mg/L for the definitive study
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (1.0 mg/L)
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
47.4 mg/L
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
2.85 mg/L
Basis for effect:
mobility
Remarks on result:
other: 95% Fiducial Limits (mg/L) 2.17 to 3.64
Duration:
48 h
Dose descriptor:
other: LC90
Effect conc.:
22.4 mg/L
Basis for effect:
mobility
Remarks on result:
other: 95% Fiducial Limits (mg/L) 14.5 to 44.7
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.63 mg/L
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
1.13 mg/L
Basis for effect:
mobility
Details on results:
The lowest concentration of test material producing 100% mortality could not be determined from this study. All concentrations of test material produced some mortality in this study. There was evidence of a dose response in this study. All parameters measured in this study lay within the acceptable tolerance limits.
Results with reference substance (positive control):
A 1.0 mg/L 3,5-dichlorophenol test immobilised 10% of the copepods at 24 hours and 90% of the copepods after 48 hours.
Reported statistics and error estimates:
See effect concentration section

Percentage immobilisation data for copepods exposed to MK92K, in definitive study STL095535

 

Test material conc. (mg/L)

Immobilisation (%)

24 hours

48 hours

0

3

10

0.63

5

15

1.13

0

45

2.00

5

50

3.56

0

55

6.32

10

70

11.25

10

85

DCP

10

90

Validity criteria fulfilled:
yes
Conclusions:
After 48 hours exposure to MK92K, the LC50 was determined to be 2.85 mg/L. The 24-hour LC50 was determined to be 47.4 mg/L. The highest concentration of test material producing No Observalbe Effect (NOEC) was 0.63 mg/L in this study. The Lowest Observed Effect Concentration (LOEC) was determined to be 1.13 mg/L.
Executive summary:

In study STL095535 a sample of MK92K was assessed for potential toxicity to the marine copepod Acartia tonsa in a static test over 48 hours according to the draft method ISO 14669:1999(E).

 

Water Accommodated Fractions containing concentrations of MK92K, were prepared over the range 0.63 mg/L to 11.25 mg/L. Acartia tonsa were exposed to the test concentrations in order to determine the concentration which killed 50% of the copepods over 48 hours (LC50). The results are summarised in the table below:

 

Time (hours)

LC50(mg/L)

95% Fiducial Limits (mg/L)

LC90(mg/L)

95% Fiducial Limits (mg/L)

NOEC (mg/L)

LOEC (mg/L)

24

47.4

*

*

-

-

-

-

-

48

2.85

2.17

3.64

22.4

14.5

44.7

0.63

1.13

*Figure not calculable, - Not applicable

 

The lowest concentration of test material producing 100% mortality could not be determined from this study. All concentrations of test material produced some mortality in this study.

 

There was evidence of a dose response in this study.

 

All parameters measured in this study lay within the acceptable tolerance limits.

Description of key information

In the marine invertebrate study, MK92K was assessed for potential toxicity to the marine copepod, Acartia tonsa, in a static test over 48 hours. After 48 hours exposure to MK92K, the LC50 was determined to be 2.85 mg/L. The 24-hour LC50 was determined to be 47.4 mg/L. The highest concentrations of test material producing No Observable Effect (NOEC) was 0.63 mg/L in this study. The Lowest Observed Effect Concentration (LOEC) was determined to be 1.13 mg/L.
In the freshwater invertebrate study, the 48-hour acute toxicity of MK92K to the freshwater planktonic crustacean, Daphnia magna, was determined. MK92K was toxic to Daphnia magna under the conditions of the test. No immobility was observed in the control treatment. The highest nominal active ingredient concentration at which no significant immobility occured was 1.97 mg/L. The lowest nominal active ingredient concentration at which 100% immobility occurred was 6.67 mg/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
3.1 mg/L
EC50/LC50 for marine water invertebrates:
2.85 mg/L

Additional information

The toxicity to the marine invertebrate, Acartia tonsa, was determined according to the draft method, ISO 14669:1999(E).

The toxicity to the freshwater invertebrate, Daphnia magna, was determined in accordance with OECD guideline 202 (Daphnia sp. Acute Immobilisation Test).