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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 April 2004 to 12 May 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
not specified
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Frequency: at t= 0h (in combination with NOTOX Project 406966) and t= 48 h.
Volume: 5ml from the approximate centre of the test vessels
Storage: Samples were stored in a freezer until analysis.
Vehicle:
no
Details on test solutions:
The standard test procedures required generation of test solutions, which should contain completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that disturb the test system shou1d be prevented (e.g. film of the test substance on the water surface).
Hatcol 5127 is a light yellow liquid. The water solubility of Hatcol 5127 at 20.4 +/- 0.7°C was determined to be < 0.14x10-3 g/l in a preliminary experiment (pH was between 8.0-8.1, NOTOX Project 366035).
AII test solutions with a loading rate at or above 1.0 mg/l were prepared separately {loading rates of 1.0, 10 and 100 mg/l). These saturated solutions were stirred for three days to reach maximum solubility. After the stirring period both the 10 and the 100 mg/l loading rates were inhomogenous emulsions with a test substance floating layer, while the 1.0 mg/l loading rate was observed to be clear and colourless. Collection of the water phase by siphoning or centrifugation was not an option considering the specific gravity of the test substance {see also NOTOX Project 388035). After the stirring period all three mixtures/solutions were therefore filtered twice. First through a paper filter (Schleicher and SchuelI 604) to remove the larger undissolved test substance particles {ca. > 5pm) and subsequently through a membrane filter
{Schleicher and Schuell RC55, 0.45pm). Finally a ten fold dilution was prepared from the filtrate prepared at a loading rate of 1.0 mg/l and indicated as 0.1 mg/l. All test solutions were clear and colourless. The test solutions originated from those used for the simultaneously performed acute fish toxicity test {NOTOX Project 406968).
Test organisms (species):
Daphnia magna
Details on test organisms:
Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.
Source: In house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality.
20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Characteristics: For the test selection of young daphnia with an age of < 24 hours, from parental daphnids of more than two weeks old.
Breeding
Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an a11-glass culture vessel.
Maximum age of the cultures: 4 weeks
Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
Temperature of medium: 18-22˚C
Feeding: Daily, a suspension of fresh water algae.
Medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).
Composition of medium M7:
ISO medium: the following chemicals (analytical grade) are dissolved in tap water which is purified by reverse osmosis (mill-RO, Millipore Corp. Bedford, Mass., USA):
Marco salts:
CaCl22H2o 293.8 mg/L
MgSO47H2O 123.3 mg/L
NaHCO3 64.8 mg/L
KCl 5.8 mg/L
Med1um M7: trace elements, macro nutrients and vitamins are added to freshly prepared ISO medium to reach the following concentrations:
B: 0.125 mg/L
Fe: 0.05 mg/L
Mn: 0.025 mg/L
U, Rb and Sr: 0.0125 mg/L
Mo: 0.0063 mg/L
Br: 0.0025 mg/L
Cu: 0.0016 mg/L
Zn: 0.0063 mg/L
Co and I: 0.0025 mg/L
Se: 0.0010 mg/L
V: 0.0003 mg/L
Na2 EDTA.2H2O: 2.5 mg/L

Macro nutrients
Na2SIO3.9H2O: 10 mg/L
NaNO3: 0.27 mg/L
KH2PO4: 0.14 mg/L
K2HPO4: 0.18 mg/L
Vitamins:
Thiamine: 75.0 µg/L
B12: 1.0 µg/L
Biotin: 0.75 µg/L
The hardness: 250 mg/1 expressed as CaCO3 and the pH: 8.0 +/- 0.2 after aeration.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
250 mg/l expressed as CaCO3
Test temperature:
18-22˚C
pH:
8.0 +/- 0.2 after aeration.
Dissolved oxygen:
Not specified
Salinity:
Not specified
Conductivity:
Not specified
Nominal and measured concentrations:
Nominal 1.0, 10.0, 100.0 mg/L
Details on test conditions:
Measurements and recordings
lmmobility (including mortality): At 24 hours and at 48 hours.
pH and dissolved oxygen: At the beginning and at the end of the test, for all concentrations and the control.
Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.

Test procedure and conditions
Test duration: 48 hours
Test type: static
Test vessels: 100mL, all glass
Medium: ISO
Number of daphnia: 20 per concentration
Loading: 5 per vessel containing 80 mL medium
Light: 16 hours photoperiod daily
Feeding: No feeding
Aeration: No aeration of the test solutions
Introduction of daphnia: Within 2 ¼ hours after preparation of the test solutions.
Reference substance (positive control):
yes
Key result
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
0.02 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.02 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
The concentrations of Hatcol 5127 were determined by Gas Chromatography with mass spectrometric detection (GC/MS). The test substance consisted of a mixture of molecules with different molecular weights, which differed in water solubility, resulting in a number of peaks in the chromatogram of the test substance solutions. It was not possible to determine which molecule was responsible for the toxicological response, if any. Furthermore, since not all components were quantifiable in the calibration chromatograms, it was not possible to determine the concentration of the total test substance. Therefore, the toxicological evaluation was based on the water-soluble fraction at the loading rate in addition, the actual concentration was estimated from the largest peak observed in the chromatograms of Hatcol 5127.
The analytical results showed that the filtered solution prepared at a loading rate of 100 mg/l contained a mean initial concentration of 0.031 mg/l. After 48 hours this concentration had decreased to 0.012 mg/l.
The average exposure concentration at a loading rate of 100 mg/l was calculated to be 0.020 mg/l. The observed decrease was probably a consequence of the extremely low solubility.

Immobility
No immobilization was observed at any of the concentrations tested.

Experimental conditions
These test conditions remained within the limits prescribed by the protocol (pH: 6.0-8.5, not varying by more than 1 unit; oxygen: >7 mg/l at the start, ≥ 5 mg/l at the end of the test).
The temperature of the test medium was 20.3°C at the start of the test. The temperature continuously measured in a temperature control vessel varied between 18.8 and 19.6°C during the test, and complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).
Results with reference substance (positive control):
The 24h-EC50 was 0.84 mg/l with a 95% confidence interval between 0.78 and 0.94 mg/l.
The 48h-EC50 was 0.66 mg/l with a 95% confidence interval between 0.54 and 0.87 mg/l.

pH and oxygen concentrations during the final test

Loading rate Hatcol 5127 (mg/L)

Start (t=0 h)

End t=48 h)

pH

O2

pH

O2

Blank-control

7.9

9.1

7.9

8.7

0.1

7.9

8.9

7.9

8.8

1.0

7.8

7.0

7.8

8.8

10

7.9

7.1

7.8

8.9

100

7.9

7.1

7.8

8.8

 

Acute immobilization of daphnia after 24 and 48 hours in the final EC50-test

 

Loading rate Hatcol 5127 (mg/L)

Vessel number

Number Daphnia exposed

Response at 24 h

Response at 48 h

Number

Total %

Number

Total %

Blank-control

A

B

C

D

5

5

5

5

0

0

0

0

0

0

0

0

0

0

0.1

A

B

C

D

5

5

5

5

0

0

0

0

0

0

0

0

0

0

1.0

A

B

C

D

5

5

5

5

0

0

0

0

0

0

0

0

0

0

10

A

B

C

D

5

5

5

5

0

0

0

0

0

0

0

0

0

0

100

A

B

C

D

5

5

5

5

0

0

0

0

0

0

0

0

0

0

 

Acute immobilization of daphnia after 24 and 48 hours in the reference test with potassium dichromate:

Concentration (mg/L)

Number exposed

% immobile

Expected response (%) after 48 hours1

24h

48h

Minimal

Maximal

Blank-control

20

0

0

0

102

0.1

20

0

0

0

10

0.18

20

0

0

0

10

0.32

20

0

5

0

30

0.56

20

0

5

0

100

1.0

20

80

100

40

100

1.8

20

100

100

100

100

1: Based on historical data of the previous years (n>60)

2: a maximum response of 10% does not invalidate the results of the test

 

Concentrations of Hatcol 5127 in test medium (final test)

Time of sampling [hours]

Date of sampling

[dd-mm-yy]

Date of pre-treatment2

[dd-mm-yy]

Date of analysis4

[dd-mm-yy]

Concentration

Loading rate [mg/L1]

Analysed [mg/L]

Relative to initial %

0

10-05-043

24-05-04

26-05-04

0

0

100

100

<0.001

<0.001

0.0338

0.0275

 

48

12-05-04

24-05-04

26-05-04

0

0

100

100

<0.001

<0.001

0.0130

0.0119

n.a.

n.a.

42

39

1: 0.45µm filtered solution

2: samples were frozen until analysis

3: Combined with NOTOX project 406968

4: Due to problems with the HPLC equipment the samples and calibration solutions were stored at ambient temperature at analysed on 26-05-04

n.a.: Not applicable

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study Hatcol 5127 induced no acute immobilisation of Daphnia magna exposed to a filtered solution prepared at a loading rate of 100 mg/l, containing an average exposure concentration of 0.020 mg/l (NOEC).
Due to the extremely low water solubility of Hatcol 5127, concentration levels that might be toxic to Daphnia magna could not be reached.
Executive summary:

 

Acute Toxicity Study in Daphnia magna with Hatcol 5127.

The study procedures described in this report were based on the ISO International Standard 6341, 1996. ln addition, the procedures were designed to meet the test methods and validity criteria of the EEC directive 92/69, Part C.2, 1992, the OECD guideline No. 203, 1992 and the OECD series on testing and assessment number 23, December 14, 2000.

Hatcol 5127 is a light yellow liquid. The water solubility of Hatcol 5127 at 20.4 +/-0.7°C was determined to be < 14.0 x 10^-3 g/l in a preliminary experiment (pH was between 8.0-8.1, NOTOX Project 388035).

AII test solutions with a loading rate at or above 1.0 mg/l were prepared separately (loading rates of 1.0, 10 and 100 mg/l). These saturated solutions were stirred for three days to reach maximum solubility. Collection of the water phase by siphoning or centrifugation was not an option considering the specific gravity of the test substance (see also NOTOX Project 388035). After the stirring period all three mixtures/solutions were therefore filtered twice. First through a paper filter to remove the larger undissolved test substance particles (ca. > 5pm) and subsequently through a membrane filter (0.45pm). Finally a ten-fold dilution was prepared from the filtrate prepared at a loading rate of 1.0 mg/l AII test solutions were clear and colourless.

A final test was performed exposing twenty daphnids per concentration to a blank-control, 0.45µm filtered solutions prepared at loading rates of 1.0, 10 and 100 mg/l and in addition to a ten-fold dilution of the filtrate prepared at a loading rate of 1.0 mg/l for a period of 48 hours.

Samples for analytical confirmation of actual exposure concentrations were taken from the blank-control and the highest test concentration at the start and at the end of the test.

Since not all components of Hatcol 5127 were quantifiable in the calibration chromatograms, analytical results were based on the largest peak. Based on this peak the filtered solution prepared at a loading rate of 100 mg/l contained a mean initial concentration of 0.031 mg/l. After 48 hours this concentration had decreased to 0.012 mg/l. The average exposure concentration at a loading rate of 100 mg/l was calculated to be 0.020 mg/l. The observed decrease after 48 hours of exposure was probably a consequence of the extremely low solubility.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

Hatcol 5127 induced no acute immobilisation of Daphnia magna exposed to a filtered solution prepared at a loading rate of 100 mg/l, containing an average exposure concentration of 0.020 mg/l (NOEC).

Due to the extremely low water solubility of Hatcol 5127, concentration levels that might be toxic to daphnia magna could not be reached.

Description of key information

Under the conditions of the present study Hatcol 5127 induced no acute immobilisation of Daphnia magna exposed to a filtered solution prepared at a loading rate of 100 mg/l, containing an average exposure concentration of 0.020 mg/l (NOEC).

Due to the extremely low water solubility of Hatcol 5127, concentration levels that might be toxic to Daphnia magna could not be reached. 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
100 mg/L

Additional information

Acute Toxicity Study in Daphnia magna with Hatcol 5127.

The study procedures described in this report were based on the ISO International Standard 6341, 1996. ln addition, the procedures were designed to meet the test methods and validity criteria of the EEC directive 92/69, Part C.2, 1992, the OECD guideline No. 203, 1992 and the OECD series on testing and assessment number 23, December 14, 2000.

Hatcol 5127 is a light yellow liquid. The water solubility of Hatcol 5127 at 20.4 +/-0.7°C was determined to be < 14.0 x 10^-3 g/l in a preliminary experiment (pH was between 8.0-8.1, NOTOX Project 388035).

AII test solutions with a loading rate at or above 1.0 mg/l were prepared separately (loading rates of 1.0, 10 and 100 mg/l). These saturated solutions were stirred for three days to reach maximum solubility. Collection of the water phase by siphoning or centrifugation was not an option considering the specific gravity of the test substance (see also NOTOX Project 388035). After the stirring period all three mixtures/solutions were therefore filtered twice. First through a paper filter to remove the larger undissolved test substance particles (ca. > 5pm) and subsequently through a membrane filter (0.45pm). Finally a ten-fold dilution was prepared from the filtrate prepared at a loading rate of 1.0 mg/l AII test solutions were clear and colourless.

A final test was performed exposing twenty daphnids per concentration to a blank-control, 0.45µm filtered solutions prepared at loading rates of 1.0, 10 and 100 mg/l and in addition to a ten-fold dilution of the filtrate prepared at a loading rate of 1.0 mg/l for a period of 48 hours.

Samples for analytical confirmation of actual exposure concentrations were taken from the blank-control and the highest test concentration at the start and at the end of the test.

Since not all components of Hatcol 5127 were quantifiable in the calibration chromatograms, analytical results were based on the largest peak. Based on this peak the filtered solution prepared at a loading rate of 100 mg/l contained a mean initial concentration of 0.031 mg/l. After 48 hours this concentration had decreased to 0.012 mg/l. The average exposure concentration at a loading rate of 100 mg/l was calculated to be 0.020 mg/l. The observed decrease after 48 hours of exposure was probably a consequence of the extremely low solubility.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

Hatcol 5127 induced no acute immobilisation of Daphnia magna exposed to a filtered solution prepared at a loading rate of 100 mg/l, containing an average exposure concentration of 0.020 mg/l (NOEC).

Due to the extremely low water solubility of Hatcol 5127, concentration levels that might be toxic to daphnia magna could not be reached.