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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
calculation (if not (Q)SAR)
Remarks:
estimated by calculation
Adequacy of study:
supporting study
Study period:
December 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Validated QSAR calculation method, using ECOSAR v1.00 model. A QPRF is attached. The substance is recognised as part of the rulebase utilised by the EPI Suite model data set, and is in model Applicability Domain. Further details can be found within the appended report below or at http://www.epa.gov/oppt/exposure/pubs/episuite.htm. This system is recognised in ECHA Guidance document CHAPTER R.6 – QSARS AND GROUPING OF CHEMICALS, Pg 47
Qualifier:
no guideline required
Principles of method if other than guideline:
The substance is a hydrocarbon UVCB of limited solubility.It is unlikely that a study result would give anything other than a “greater than” limit value of the highest value available in the test. This endpoint is therefore characterized using quantitative structure property relationships for representative hydrocarbon structures that could be present within this UVCB substance. 70 proposed molecules are assessed, in order to provide a suitable range of likely values associated with the substance. Details on the US EPA On-Line EPI Suite™ ECOSAR v1.00 programme are detailed below.
GLP compliance:
no
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):Not applicable - QSAR assessment.
Analytical monitoring:
not required
Details on sampling:
Not applicable - QSAR assessment.
Vehicle:
no
Details on test solutions:
Not applicable - QSAR assessment.
Test organisms (species):
other: Not applicable - QSAR assessment.
Details on test organisms:
Not applicable - QSAR assessment.
Test type:
other: Not applicable - QSAR assessment.
Water media type:
not specified
Limit test:
yes
Post exposure observation period:
Not applicable - QSAR assessment.
Hardness:
Not applicable - QSAR assessment.
Test temperature:
Not applicable - QSAR assessment.
pH:
Not applicable - QSAR assessment.
Dissolved oxygen:
Not applicable - QSAR assessment.
Salinity:
Not applicable - QSAR assessment.
Nominal and measured concentrations:
Not applicable - QSAR assessment.
Details on test conditions:
Not applicable - QSAR assessment.
Reference substance (positive control):
not required
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
>= 0 - <= 0.002 mg/L
Nominal / measured:
estimated
Conc. based on:
not specified
Basis for effect:
mortality
Remarks on result:
other: 70 specimen structures were selected for assessment. The values presented are above the limit of solubility in water.
Details on results:
The substance is a hydrocarbon UVCB of limited solubility.It is unlikely that a study result would give anything other than a “greater than” limit value of the highest value available in the test. This endpoint is therefore characterized using quantitative structure property relationships for representative hydrocarbon structures that could be present within this UVCB substance. 70 proposed molecules have been assessed, in order to provide a suitable range of likely values associated with the substance. These results, upon first inspection, indicate that the classification as “toxic” could apply. However, if the log Kow is greater than 5.0, or if the LC50 exceeds the water solubility by 10X, no effects at saturation are predicted. Given that the substance is insoluble in water, and taken in conjunction with the acute toxicity to Daphnia study results, it is clear that the substance does not demonstrate any effects at the limit of solubility within water. This is based on the fact that this category of substances does not demonstrate any significant toxicity to aquatic organisms, nor does it contain significant impurities that could effect the outcome of the a specific study. On this basis, it is proposed that the substance is not harmful to fish at the limit of solubility within water.
Results with reference substance (positive control):
Not applicable - QSAR assessment.
Reported statistics and error estimates:
Not applicable - QSAR assessment.

Tabulated data from evaluation of various potential structures present in NovaSpec 450 Base Oil

No

Derivation

SMILES Code

Molecular Formula

MW

Acute toxicity to Fish (96 h LC50) mg/l

1

M

CC(C)CCCC(C)CCCC(C)CC

C15H32

212.41

0.002

2

M-C8

CC(C)C(CCC(C)CCCC(C)CC)C(CC)CCCCC

C23H48

324.63

1.75E-06

3

M-C10

CC(C)C(CCC(C)CCCC(C)CC)C(CCCC)CCCCC

C25H52

338.65

2.59E-07

4

M-C12

CC(C)C(CCC(C)CCCC(C)CC)C(CCCCC)CCCCCC

C27H56

380.73

3.83E-08

5

M-C14

CC(C)C(CCC(C)CCCC(C)CC)C(CCCCCCCC)CCCCC

C29H60

408.79

5.62E-09

6

M-C16

CC(C)C(CCC(C)CCCC(C)CC)C(CCCCCCCCCC)CCCCC

C31H64

436.84

8.21E-10

7

M-C8

CCC(CCCCC)C(CCC(C)C)C(C)CCCC(C)CC

C23H48

324.63

1.75E-06

8

M-C8

CCC(CCCCC)C(CCC(C)CC)C(C)CCCC(C)C

C23H48

324.63

1.75E-06

9

M-C10

CC(CCCC(C)CCCC(C)CC)CC(CCCCC)CCCC

C25H52

338.65

2.24E-07

10

M-C10

CC(CCCC(C)CC)C(CCC(C)C)C(CCCCC)CCCCCC

C25H52

338.65

3.83E-08

11

M-C12

CC(CCCC(C)CC)C(CCC(C)C)C(CCCCC)CCCCCC

C27H56

380.73

3.83E-08

12

M-C12

CC(CCCC(C)C)C(CCC(C)CC)C(CCCCCC)CCCCC

C27H56

380.73

3.83E-08

13

M-C12

CC(CC)C(CCC(C)CCCC(C)C)C(CCCCC)CCCCCC

C27H56

380.73

3.83E-08

14

M-C12

CC(CCCC(C)C)C(CCC(C)CC)C(CCCC)CCCCCCC

C27H56

380.73

3.83E-08

15

M-C14

CC(CC(CCCCCC)CCCCCCC)CCCC(C)CCCC(C)CC

C29H60

408.79

4.84E-09

16

M-C14

CC(CCCC(C)CC)C(CCC(C)C)C(CCCCC)CCCCCCCC

C29H60

408.79

5.62E-09

17

M-C14

CC(CC)C(CCC(C)CCCC(C)C)C(CCCCC)CCCCCCCC

C29H60

408.79

5.62E-09

18

M-C14

CC(CCCC(C)CC)C(CCC(C)C)C(CCCCCCCCC)CCCC

C29H60

408.79

5.62E-09

19

M-C14

CC(CCCC(C)C)C(CCC(C)CC)C(CCCCCCC)CCCCCC

C29H60

408.79

5.62E-09

20

M-C16

CC(CCCC(C)CCCC(C)CC)CC(CCCCCCCC)CCCCCCC

C31H64

436.84

7.07E-10

21

M-C16

CC(C)CCC(C(CCCCCCCC)CCCCCCC)C(C)CCCC(C)CC

C31H64

436.84

8.21E-10

22

M-C16

CC(C)C(CCCCCCCCCCCCCCCC)CCC(C)CCCC(C)CC

C31H64

436.84

7.07E-10

23

M-Farn

CC(CC(CCC(C)CCCC(C)CC)C(C)C)CCCC(C)CCCC(C)CC

C30H62

422.81

2.89E-09

24

M-Farn

CC(C)CCCC(CCCC(C)CC)CC(CCC(C)CC)C(C)CCCC(C)C

C30H62

422.81

2.89E-09

25

M-Farn-C8

CC(C)CCCC(C)CCC(CC(CCCC(C)CC)CCC(C(CC)CCCCC)C(C)C)C(C)CC

C38H78

535.03

1.72E-12

26

M-Farn-C8

CC(CCCC(C)CCCC(C)CCCC(C)CCCC(C)CC)C(CCC(C)C)CCCCCCCC

C38H78

535.03

1.28E-12

27

M-Farn-C10

CC(C)CCCC(C)CCC(CC(CCCC(C)C(CCC(C)C)C(CCCCCCC)CC)CC)C(C)CC

C40H82

563.08

2.48E-13

28

M-Farn-C12

CC(CCCC(C)C)C(CCC(CC(CCC(C)CCCC(C)C)C(C)CC)CC)C(CCCCC)CCCCCC

C42H86

591.13

3.56E-14

29

M-Farn-C14

CC(C)C(CCC(C)CCCC(C)CC)C(CC(C)CCCC(C)CC)C(C(C)C)C(CCCC)CCCCCCCCC

C44H90

619.18

5.91E-15

30

M-Farn-C16

CC(CC(CCCC)CCCCCCCCCCC)CCCC(CCCC(C)CC)CC(CCC(C)C)C(C)CCCC(C)CC

C46H94

647.24

6.27E-16

31

M-C8-C12

CCCCCC(CC(CC)CCCC(C)C(CCC(C)C)C(CCCCC)CC)CCCCCC

C35H72

492.95

2.01E-11

32

M-C8-C14

CCCC(CCCCCCCCCC)CC(CCC(CCCCCCCC)C(C)C)CCCC(C)CC

C37H76

520.99

2.50E-12

33

M-C8-C16

CC(CCCC(C)CC)CCCC(CCCCCCCCC)CC(CCCC)CCCCCCCCCCC

C39H80

549.05

3.10E-13

34

M-C10-C8

CCCCC(CCCCC)C(CCC(CC(C)CCCCCC)CCCC(C)CC)C(C)C

C33H68

464.89

1.39E-10

35

M-C10-C12

CCC(CCCCCCC)C(CCC(C)CC)C(C)CCCC(C)CC(CCCCCC)CCCCC

C37H76

520.99

2.90E-12

36

M-C10-C16

CCC(CCCCCCC)C(CCC(C)CC)C(C)CCCC(C)CC(CCCCC)CCCCCCCCCC

C41H84

577.11

6.01E-14

37

M-C12-C8

CC(CCCCCC)CC(CC)CCCC(C)C(CCC(C)C)C(CCCCC)CCCCCC

C35H72

492.95

2.01E-11

38

M-C12-C14

CC(CC(CCCCCCCC)CCCCC)CCC(C(CCCCC)CCCCCC)C(C)CCCC(C)CC

C41H84

577.11

6.01E-14

39

M-C12-C16

CCCCCC(CC(CCCC(C)CC)CCCC(C)CCCCCCCCCCCCC)CCCCCCCCCC

C43H88

605.16

6.39E-15

40

M-C14-C8

CC(C)C(CCC(CCCC(C)CC)CC(C)CCCCCC)C(CCCCC)CCCCCCCC

C37H76

520.99

2.90E-12

41

M-C14-C10

CCCCC(CCCCCCCCC)C(CCC(C)CC)C(CC(C)CCCCCCCC)CCCC(C)C

C39H80

549.05

4.18E-13

42

M-C14-C16

CCCCCCCCCCCC(CCCC)CC(CCCC(C)CC)CCCC(C)CC(CCCCCCCC)CCCCC

C45H92

633.21

1.06E-15

43

M-C16-C8

CC(C)C(CCC(CC(C)CCCCCC)CCCC(C)CC)C(CCCCCCCCCC)CCCCC

C39H80

549.05

4.18E-13

44

M-C16-C16

CC(CC(CCCCCCCCCC)CCCCC)C(CCC(C)CCCC(C)CC)CCCCCCCCCCCCCCCC

C47H96

661.26

1.52E-16

45

M-Farn-Farn (Deriv 1)

CC(C)CCCC(C)CCCC(CC)CC(CCC(C)CC)C(C)CCCC(C)CC(CCC(C)CC)C(C)CCCC(C)C

C45H92

633.21

2.59E-15

46

M-Farn-Farn (Deriv 2)

CC(CCCC(C)CCCC(C)CCCC(C)CC)C(CC(C)CCCC(C)CCCC(C)CC)CCC(C)CCCC(C)C

C45H92

633.21

2.23E-15

47

M-Farn-Farn (Deriv 3)

CC(CC(CCC(C)CCCC(C)CC)C(C)CC(CCC(C)CCCC(C)CC)C(C)C)CCCC(C)CCCC(C)CC

C45H92

633.21

2.59E-15

48

M-C8-C8-C8

CC(C)C(CCC(CC(C)CCCCCC)CCC(CCCCCCCC)C(C)CC)C(CC)CCCCC

C39H80

549.05

4.85E-13

49

M-C10-C10-C10

CC(CC(CC(C)CC)CCCCCCCCCC)C(CCCCCCCCCC)CCC(C)CCCCCCCCCCC

C45H92

633.21

1.06E-15

50

M-C12-C12-C12

CC(C(C)CCCC(CCCC(C)CC(C)CCCCCCCCCC)CC(C)CCCCCCCCCC)C(CCCCCC)CCCCC

C51H104

717.37

4.14E-18

51

M-C14-C14-C14

CCCCCC(CC(CCCC(C)C(CCC(C)CCCCCCCCCCCCCCC)C(CCCCCCCC)CCCCC)CC)CCCCCCCC

C57H116

801.53

1.02E-20

52

M-C16-C16-C16

CCCCCCCCCC(CC(CCCC(C)C(CCCCCCCCCCCCCCCC)CCC(C)CC(CCCCCCC)CCCCCCCC)CC)CCCCCC

C63H128

885.69

2.87E-23

53

M-Farn-Farn-C8

CC(CCCC(CC(CCCCC)CC)C(CCC(C)C)C(CCC(C)C)C(C)CCCC(C)CC)CCCC(CCCC(C)C)CCCC(C)CC

C53H108

745.42

1.24E-18

54

M-Farn-Farn-C10

CC(CCCC(C)CC(CC(CC(C)CCCC(C)CCCC(C)CC)C(C)C(C)CCCCCCCCCC)C(C)CCCC(C)C)CCCC(C)CC

C55H112

773.48

1.76E-19

55

M-Farn-Farn-C12

CC(CCCC(C)C)C(CCC(C)CC)CC(CCCC(C)CCC(CC(C)CCCC(C)CCCC(C)CC)C(C)CC)CC(CCCCCC)CCCCC

C57H116

801.53

2.49E-20

56

M-Farn-Farn-C14

CC(CC(CCC(C)CCC(CCCC(C)C)CCCC(C)CC)C(C)CCCC(C)CC)C(CCC(C)CCCC(C)CC)C(CCCCCCC)CCCCCC

C59H120

829.58

3.52E-21

57

M-Farn-Farn-C16

CC(C)CCCC(CCCC(C)CC)CCC(CCCC(C)CCC(CCCCCCCCC)CCCCCC)C(CC(CCCC(C)CC)CCCC(C)C)CCC(C)C

C61H124

857.64

4.28E-22

58

M-Farn-C8-C10

CC(CCCC(C)CC)CCCC(C)CCC(CCCC(C)CCCC(C)C(C)CCCCCCCCCC)CCCCCCCCC

C48H98

647.24

7.71E-17

59

M-Farn-C10-C14

CC(CCCC(C)CC)CCCC(CCC(CCCC(C)CCCC(C)CCCCCCCCCCC)CC)CC(CCCC)CCCCCCCCC

C54H110

759.45

2.21E-19

60

M-Farn-C14-C16

CCCCCCCCC(CCCCC)C(CCC(C)C)C(C)CCCC(CCC(CC)C(CCC(C)CCCC(C)C)C(CCCCCCC)CCCCCCCC)CC

C60H112

843.61

9.82E-22

61

M-Farn-C16-C16

CC(CCCC(C)CC)C(CCC(C)CCC(CC(CC)CCCCCCCCCCCCC)CCCC(C)CCCC(C)CC)CCCCCCCCCCCCCCCC

C62H126

871.66

1.03E-22

62

M-Farn-C8-C16

CCCC(CCCC)C(CCC(C)CC)C(CCC(CCCC(C)CC)CCCC(C)CC(CCCCCCCCCC)CCCCC)CCCC(C)C

C54H110

759.45

2.98E-19

63

M-C8-C10-C12

CC(CCCC(C)CCCC(CC(CCCCC)CC)C(C)C(CC)CCCCCCC)CCCCCCCCCCCCC

C45H92

633.21

1.23E-15

64

M-C12-C14-C16

CCCCC(CC(CCCC(C)C(C)C(CCCCCC)CCCCCCCCC)CCCC(C)CCCCCCCCCCCCC)CCCCCCCCC

C57H116

801.53

1.02E-20

65

M-C8-C12-C16

CC(C(CC(C)CCCCCCCCCC)CCCC(C)CCCC(C)CC(CCCCCCCC)CCCCCCC)C(CCCCC)CC

C51H104

717.37

4.14E-18

66

M-C10-C12-C14

CCCCC(CCCCCCC)C(CCC(C)CCC(CCCCC)CCCC)C(C)CCCC(C)CC(CCCCCC)CCCCCCC

C51H104

717.37

4.14E-18

67

M-C8-C14-C16

CC(CCC(CCCCCCCC)C(C)CCCC(C)CCC(C)CCCCCCCCCCCC)CC(CCCCCCCCC)CCCCCC

C53H108

745.42

5.06E-19

68

M-Farn-Farn-Farn (Deriv 1)

CC(CC(CCC(C)CC)C(CC(CCC(C)CCCC(C)CC)C(C)C)CCCC(C)CCC(CCCC(C)CCCC(C)C)CC)CCCC(C)CCCC(C)CC

C60H112

843.61

1.78E-21

69

M-Farn-Farn-Farn (Deriv 2)

CC(C)C(CCC(C)CCCC(C)CC)CC(C)C(CCC(C)C(CC(C)CCCC(C)CCCC(C)CC)CCC(C)CC)C(CCC(C)CCCC(C)CC)C(C)C

C60H112

843.61

2.40E-21

70

M-Farn-Farn-Farn (Deriv 3)

CC(CC)CCCC(C)CCCC(C)CCC(CCCC(C)CC(C)C(C)CCCC(C)C(CCC(C)C)C(CCC(C)C)C(C)CCCC(C)CC)CCCC(C)CC

C60H112

843.61

2.07E-21

 

Validity criteria fulfilled:
yes
Conclusions:
The substance is a hydrocarbon UVCB of limited solubility.It is unlikely that a study result would give anything other than a “greater than” limit value of the highest value available in the test. This endpoint is therefore characterized using quantitative structure property relationships for representative hydrocarbon structures that could be present within this UVCB substance. 70 proposed molecules have been assessed, in order to provide a suitable range of likely values associated with the substance. These results, upon first inspection, indicate that the classification as “toxic” could apply. However, if the log Kow is greater than 5.0, or if the LC50 exceeds the water solubility by 10X, no effects at saturation are predicted. Given that the substance is insoluble in water, and taken in conjunction with the acute toxicity to Daphnia study results, it is clear that the substance does not demonstrate any effects at the limit of solubility within water. This is based on the fact that this category of substances does not demonstrate any significant toxicity to aquatic organisms, nor does it contain significant impurities that could effect the outcome of the a specific study. On this basis, it is proposed that the substance is not harmful to fish at the limit of solubility within water.
Executive summary:

The substance is a hydrocarbon UVCB of limited solubility.It is unlikely that a study result would give anything other than a “greater than” limit value of the highest value available in the test. This endpoint is therefore characterized using quantitative structure property relationships for representative hydrocarbon structures that could be present within this UVCB substance. 70 proposed molecules have been assessed, in order to provide a suitable range of likely values associated with the substance.

These results, upon first inspection, indicate that the classification as “toxic” could apply. However, if the log Kow is greater than 5.0, or if the LC50 exceeds the water solubility by 10X, no effects at saturation are predicted.

Given that the substance is insoluble in water, and taken in conjunction with the acute toxicity to Daphnia study results, it is clear that the substance does not demonstrate any effects at the limit of solubility within water. This is based on the fact that this category of substances does not demonstrate any significant toxicity to aquatic organisms, nor does it contain significant impurities that could effect the outcome of the a specific study. On this basis, it is proposed that the substance is not harmful to fish at the limit of solubility within water.

No classification and labelling is proposed.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 July 2014 to 18 July 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD, EU and US EPA test guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Qualifier:
according to
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):No further information detailed in the study report.
Analytical monitoring:
no
Details on sampling:
Since the test item is poorly soluble in water and due to this there is no suitable analytical method for its determination the experiment was carried out without analytical measurements based on agreement with the Sponsor.
Vehicle:
not specified
Details on test solutions:
Because the test item is very poorly soluble in water, a test solution was prepared using a saturated solution method (water accommodated fraction, WAF) according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23. A supersaturated test item solution (nominal loading rate of 100 mg/L) was prepared by dispersing/dissolving the amount of test item into the test medium (aquarium water) two days before the start of the study. This solution was shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at approximately 20°C. The non-dissolved test material was removed by filtration through a fine (0.22 μm) filter to give the 100 % v/v saturated solution. As only limit test was carried out, further dilution of stock solution was not performed.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Species: Zebra-fish (Brachydanio rerio) Source: “Black Molly” Fish Farm, Veszprém, Hungary Justification of species: Zebra-fish (Brachydanio rerio) is one of the convenient species for acute fish toxicity test and it is recommended by the test guidelines. Number of animals: 7 fish in test concentration and control groups Body length of animals: 3.0 – 3.5 cm Acclimatisation: > 12 days Animal health: Fish were bred in a well-known fish farm, under disease- and parasite-controlled conditions. Fish were observed during the acclimatisation and test periods, the fish were healthy. There was no mortality of the population for seven days before the test.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
No post exposure observation period required for this study.
Hardness:
The hardness of the dilution water was determined as 246 mg/L (as CaCO3).
Test temperature:
The test temperature was between 20.6 and 20.9 °C.
pH:
The pH was in the range of 6.33 – 7.01.
Dissolved oxygen:
The dissolved oxygen concentration was in the range of 82 – 99 % of the air saturation value at the temperature used.
Salinity:
Not applicable - freshwater study.
Nominal and measured concentrations:
Nominal concentrations. Preliminary Range-Finding Test: 01, 1, 10, 100 % v/v saturated solutions.Definitive Test: 100% v/v saturated solution.
Details on test conditions:
The water temperature, oxygen concentration and pH were measured daily during the experiment in each test aquarium.DESCRIPTION OF THE TEST PROCEDURE The test duration was 96 hours. One aquarium was used for test group and control group, respectively. Each aquarium comprised 7 fish and 5 L test solution. The animals were not fed during the test. The loading of the test aquaria was less than 1.0 g fish/L test solution at the start of the experiment. The choice of the test concentrations was done on the basis of the results of the preliminary range finding test.OBSERVATIONS The observations of fish were carried out in the following intervals: at 3, 6, 24, 48, 72 and 96 hours. Mortality or any sub-lethal effect was not observed during the study. The test conditions (pH, temperature, oxygen saturation) were examined daily during the test. The body weights of the fish were recorded before the introduction of fish. The body weight of 7 fish per aquarium was registered. The loading of the aquaria was calculated on the basis of these body weights (g fish/litre testing liquid).
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 other: mg/L loading rate WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
> 100 other: mg/L loading rate WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 other: mg/L loading rate WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
> 100 other: mg/L loading rate WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
Preliminary Range-Finding Test A preliminary concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations can be selected for use in the definitive test. Four fish were exposed to each concentration of the test item plus controls, for 96 hours under static conditions. During the formulation procedure the test solutions were prepared individually. Test item concentrations in the Definitive Test Because no toxic effect was observed at the solubility level of the test item in the preliminary test, only this test concentration (100 % v/v saturated solution) at the solubility level of the test item in the test medium and one control group were tested in a limit test in the definitive study under static conditions.VALIDITY The achievable validity criteria were within acceptable limits and therefore the study can be considered as valid.MORTALITY DATANo sublethal effect was observed.BODY WEIGHTThere was no considerable difference observed concerning body weights between the groups.
Results with reference substance (positive control):
Reference substance not used in this study.
Reported statistics and error estimates:
Since the test item is poorly soluble in water and due to this there is no suitable analytical method for its determination the experiment was carried out without analytical measurements based on agreement with the Sponsor.

The concentration levels used and results (96 h) of the preliminary range-finding test are summarised in the following table:

Results of the Preliminary Range-Finding Test

Nominal concentrations

[% v/v saturated solution]

Untreated control

0.1

1

10

100

Number of treated animals

4

4

4

4

4

Number of dead animals

0

0

0

0

0

 

Cumulative mortality data in the Limit Test

Test Group

Cumulative Mortality (number of dead fish)

 [initial population = 7 fish]

3h

6h

24h

48h

72h

96h

Control

0

0

0

0

0

0

100 % v/v saturated solution

0

0

0

0

0

0

 

Measured and calculated data of body weight

Test Group

Measured weight of 7 fish (g)

Calculated mean weight of 1 fish (g)

Loading of testing aquarium

(g fish/L testing liquid)

Control

2.63

0.38

0.53

100 % v/v saturated solution

3.00

0.43

0.60

 

Validity criteria fulfilled:
yes
Conclusions:
The 24h, 48h, 72h and 96h LC50 value: > 100 mg/L loading rate WAF The 96h LC100 value: > 100 mg/L loading rate WAF The 96h NOEC: 100 mg/L loading rate WAF The 96h LOEC: > 100 mg/L loading rate WAF Based on the results of this study, the test item NovaSpec EL46 had no toxic effect at saturation on fish; the EC50 results and the LOEC are higher than the solubility level of the test item in the test medium.
Executive summary:

The objective of this study was to determine the median lethal concentration (LC50 value) of the test item in acute toxicity test on Zebra-fish (Brachydanio rerio).

Fish were exposed in a static test to aqueous test media, containing the test item at one concentration to demonstrate that the test item has no influence on test animals up to this test concentration.

The test method of application and the test species Zebra-fish (Brachydanio rerio) are recommended by the test guidelines.

BASIS OF STUDY:

– OECD Guideline for Testing of Chemicals, No. 203, "Fish, Acute Toxicity Test", adopted July 17, 1992.

– EPA Health Effects Test Guidelines, OCSPP 850.1075.

– Commission Regulation (EC) No 761/2009 of 23 July 2009 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACh), Annex Part C, C.1 (published in the Official Journal of the European Union L 220 of 24 August 2009).

 

The acute toxicity of NovaSpec EL46 was assessed with acute fish toxicity test on Zebra-fish (Brachydanio rerio), over an exposure period of 96 hours in a static system.

Because no toxic effect was observed at the solubility level of the test item in the preliminary test, only this test concentration (100% v/v saturated solution) at the solubility level of the test item in the test medium and one control group were tested in a limit test in the definitive study.

One aquarium was used in the test groups and one in the control groups. Each aquarium comprised seven fish and five litre test solution.

Since the test item is poorly soluble in water and due to this there is no suitable analytical method for its determination the experiment was carried out without analytical measurements based on agreement with the Sponsor.

All achievable validity criteria were met during this study.

 

Under the conditions of this acute fish toxicity study on Zebra-fish (Brachydanio rerio) the observed and calculated endpoints for the effect of NovaSpec EL46 were the following:

The 24h, 48h, 72h and 96h LC50 value: > 100 mg/L loading rate WAF

The 96h LC100 value: > 100 mg/L loading rate WAF

The 96h NOEC: 100 mg/L loading rate WAF

The 96h LOEC: > 100 mg/L loading rate WAF

Based on the results of this study, the test item NovaSpec EL46 had no toxic effect at saturation on fish; the EC50 results and the LOEC are higher than the solubility level of the test item in the test medium.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 24, 2014 to December 2, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OCED & Chinese test guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Qualifier:
according to
Guideline:
other: Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals, effects on Biotic Systems, 203 Fish Acute toxicity test [M]. China Environmental Science Press. 2013: 30~36.
Qualifier:
according to
Guideline:
other: China National Technical Committee for Standardization of Dangerous Chemicals Management. GB/T 27861-2011, Chemicals Fish Acute toxicity test [S]. Beijing;China Standards Press, 2012.
Qualifier:
according to
Guideline:
other: State Environmental Protection Administration of China. HJ/T 153-2004. The Guidelines for the test of chemical [S]. Beijing: China Environmental Science Press.2004.
Qualifier:
according to
Guideline:
other: Standard Practice for Aquatic Toxicity Testing of Lubricants: Sample Preparation and Results Interpretation. Designation: D6081 -98 (Reapproved 2009).
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):Physicochemical properties Melting point: -39 °C at 101.3 kPa Boiling point: 216 °C at 101.3 kPa Volatility: Non-volatile Vapour Pressure: 1.22 kPa at 37.8 °C Solubility in Water: Using the US EPA On-Line EPI Suite™ WSKOW v1.42 model, the water solubility range is predicted to be 2.64E-27 to 0.004421mg/L. This indicates that the substance is insoluble, with water solubilities much lower than 0.1mg/L. Solubility in Solvent Water: none Density: 0.82 Odor: None Date of expiration: July, 2016
Analytical monitoring:
yes
Details on sampling:
50mL test solutions were accurately taken and poured into the separating funnel, then 20mL, 15mL, 10mL hexyl hydride were added respectively to extract for three times (5min for each time). After that, extractions were collected and transferred into a 50mL volumetric flask and then diluted with hexyl hydride to 50mL. The actual concentrations of test substance in extractions were measured. The samples were analyzed immediately. Otherwise the samples (hexyl hydride extractions) were stored at 4°C until analyzed during one week.
Vehicle:
no
Details on test solutions:
An amount of test substance (0.500g) was obtained in a flask with a small amount of test water then transferred into 5L-jar and diluted with test water to 5000mL. And the test suspension was stirred with a magnetic stirrer for about 24h (the depth of vortex was maintained in the range of 2cm-3cm) then the nominal loading rate of 100 mg/L suspension was obtained. After the stirring was stopped, the mixture was allowed to stand for about 1 hour. Then the aqueous phase form the middle and bottom layer was removed by siphoning into another 5L-jar (the first approximated 100-200mL discarded) to give the nominal loading rate of 100 mg/L WAF (Water-accommodated fraction).
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Species: Zebra fish (Danio rerio) Reason for selection of species: The test species is recommended by "The Guidelines for the Testing of Chemicals". Supplier: Guangzhou Yangshi Breeding Farm Acclimation: Zebra fish (Danio rerio) were obtained on August 27, 2014. The batch number was FDr20140827-1. Before the start of the test, Zebra fish (Danio rerio) were acclimated for 89 days by flow-through under the same conditions (water quality: dechlorinated tap water; temperature: 23°C ± 2°C; photoperiod: 14h light / 10h dark). The dissolved oxygen concentration during acclimation was kept at least 80 % of the air saturation value. The test fish were fed at least three times per week until 24 hours before the start of the test. The mortality was 0% during 7 days before the start of the test, which met the requirement. Sensitivity Test of K2Cr2O7 to Zebra fish (Danio rerio) : Sensitivity Test of K2Cr2O7 with Zebra fish (Danio rerio) (The test report No. is 2014GST0015) showed that the 24h-LC50 of the reference substance (K2Cr2O7) was 275mg/L to Zebra fish (Danio rerio) and the 95% confidence limits ranged from 239mg/L to 322mg/L. The results were kept at the value ranging from 200mg/L to 400mg/L, which met the criterion for the validity of the test.Total number of test fish: 20 fish (The sex of fish was not determined.) Allocation to the test groups: Zebra fish (Danio rerio) were randomly picked out and allocated to each test group at random within 30 minutes.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
No post exposure observation period specified in the study report.
Hardness:
The hardness of test water was 118mg/L (expressed as CaCO3).
Test temperature:
Water temperature 22.3ºC - 23.9ºC
pH:
The test was performed without adjustment of pH and the pH was 7.74 - 8.00 during the test period.
Dissolved oxygen:
The test solutions were not aerated during the exposure. The dissolved oxygen concentration in the test solutions during the test was kept at least 60 % of the air saturation value.
Salinity:
Not applicable - Freshwater study
Nominal and measured concentrations:
The actual concentrations of 100 mg/L loading rate WAF were measured at 0h, 72h (new solution) and 24h, 96h (old solution) by GC. The blank control was measured at 0h (new solution) and 24h (old solution).According to the results of other tests, a nominal loading rate of 100mg/L WAF was selected
Details on test conditions:
Exposure conditions Type of test: The measured concentrations of the test substance in the solution were all lower than the limit of detection (LOD=0.863mg/L) during 24h, and the stability of test substance in test system could not be confirmed, so the test was conducted as semi-static test at 24h intervals. Exposure duration: 96hTest concentration: According to the results of other tests, a nominal loading rate of 100mg/L WAF was selected. Control: A blank control (test water without test substance) was set in parallel to the test concentration. Replicate: No replicates Number of fish: 10 fish / test group, 20 fish in total Volume of test solution: 4L / test vessel Loading of test fish: 10 fish / test vessel, 0.421 g fish / L Conditions of test environment Water temperature: 22.3°C - 23.9°C Dissolved oxygen concentration: The test solutions were not aerated during the exposure. The dissolved oxygen concentration in the test solutions during the test was kept at least 60 % of the air saturation value. pH: The test was performed without adjustment of pH and the pH was 7.74 - 8.00 during the test period.Light: Artificial light of white fluorescent lamp was used. The photoperiod was 14h light / 10h darkness. At the beginning and at the end of the test, the intensities of illumination of test environment were 113lx and 116 lx on average, respectively. Feeding: The fish were not fed during the exposure. Observation and measurements Vortex depth and appearance measurements: The vortex depth was measured at the start and end of the mixing period and the appearance of vortex also recorded. Observations on test substance solubility: At the start and end of each mixing period, and after the 1-Hour settlement period the appearance of the WAF was observed and recorded. After siphoning, the WAF solution was inspected under microscope.Observation of test solutions appearance: The changes of color and appearance of the test suspension were recorded daily. Measurement of light illumination: The intensity of illumination was measured at the beginning and the end of the test. Observation of test organisms: Mortalities and visible abnormalities of the test fish were recorded at 24h, 48h, 72h and 96h after the start of the test during test period. The dead fish were removed as soon as they were found. A fish is considered dead if there is no visible movement (e.g. gill movements) and if touching of the caudal peduncle produces no reaction. Measurement of temperature, pH and dissolved oxygen: The temperature, pH and dissolved oxygen of each test group were measured at 0h, 96h and 24h, 48h, 72h (before and after the renewal) after the start of the test. Measurements of pH were carried out with a portable pH meter. Measurements of temperature and dissolved oxygen were carried out with an oxygen meter. And the temperature of test water was continuously monitored during the test exposure by dual thermometer. Body weight and total length of test fish: The body weight and total length of 10 test fish in blank control were measured at the end of the test. Measurement of actual concentrations of test substance: The actual concentrations of 100 mg/L loading rate WAF were measured at 0h, 72h (new solution) and 24h, 96h (old solution) by GC. The blank control was measured at 0h (new solution) and 24h (old solution).
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 100 other: mg/L nominal loading rate WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LL0
Effect conc.:
100 other: mg/L nominal loading rate WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LL100
Effect conc.:
> 100 other: mg/L nominal loading rate WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
Vortex depth and appearance measurements: The vortex depth was recorded at the start and end of each mixing period. And the vortex was observed to be a dimple at the water surface on each occasion at the start and end of each mixing period. Observations on test substance solubility: At the start and end of each mixing period, the nominal loading rate of 100mg/L test suspension was observed to be water column with oily globules of test substance floating on the water surface. After 24h stirring and 1-hour standing period, the nominal loading rate of 100mg/L test suspension was observed to remain a clear colorless water column in the mid-depth but also with oily globules of test substance floating on the water surface. After siphoning, the microscopic inspection of WAFs showed no micro-dispersions or undisssolved test substance to be present. Observation of test solutions: The blank control and the 100mg/L loading rate WAF test solutions were observed to be clear and colorless during the test period. Intensity of illumination: At the start of the test, the intensity of illumination of test environment was 116 lx, 120 lx and 104 lx (113 lx on average). At the end of the test, the intensity of illumination of test environment was 121 lx, 117 lx and 109 lx (116 lx on average). Mortality: At the end of the test, the cumulative mortalities of blank control and 100mg/L loading rate WAF were 0%. Observed abnormal response: No abnormal responses were observed in blank control and the 100mg/L loading rate WAF.Measurement of temperature, pH and dissolved oxygen of test suspension: During the test, the measured value of pH was in the range of 7.74 to 8.00 and the dissolved oxygen was in the range of 88% to 99%. The temperature of test environment water ranged from 22.3°C to 23.9°C. The dissolved oxygen concentrations were kept not less than 60 percent of the air saturation value, which met the criterion for the validity of the test. The body weight and total length of test fish: The body weight and total length of 10 test fish in blank control were measured when the test was finished. The average and standard deviation of test fish body weight and total length were respectively 0.168g ± 0.023g and 27.36 mm ± 1.49 mm. Determination of test substance concentrationSample pre-treatment and preservation: 50mL test solutions were accurately taken and poured into the separating funnel, then 20mL, 15mL, 10mL hexyl hydride were added respectively to extract for three times (5min for each time). After that, extractions were collected and transferred into a 50mL volumetric flask and then diluted with hexyl hydride to 50mL. The actual concentrations of test substance in extractions were measured. The samples were analyzed immediately. Otherwise the samples (hexyl hydride extractions) were stored at 4°C until analyzed during one week. Analytical measurements: The measured concentrations of test substance in blank control were lower than the limit of detection (LOD = 0.863 mg/L) at 0h in the new solution and at 24h in the old solution after the start of the test. The measured concentrations of test substance in 100mg/L loading rate WAF were lower than the limit of detection (LOD = 0.863 mg/L) at 0h, 72h (new solution) and 24h, 96h (old solution) after the start of the test. Median Lethal Loading (LL50): At the end of the test, the cumulative mortality of the nominal loading rate of 100mg/L test group was 0%, so the 96h-LL50 of test substance to Zebra fish (Danio rerio) was greater than 100mg/L nominal loading rate WAF. Validity of test (1) The mortality of the control was less than 10% at the end of the test. (2) The dissolved oxygen concentration in the test solutions was kept at least 60 percent of the air saturation value throughout the test.
Results with reference substance (positive control):
Reference substance not used in this study.
Reported statistics and error estimates:
Cumulative mortalities of each group at 24h, 48h, 72h and 96h after the start of the test were calculated. The LL50 values at 24h, 48h, 72h and 96h after the start of the test were calculated by Binomial Distribution methods (CT-TOX Multi-Method Program, IBM PC Version 1.1).

Vortex depth and observation measurements at the start and end of mixing period

Measurements

100 mg/L loading rate WAF test group

0h

24h

48h

72h

Start

End

Start

End

Start

End

Start

End

Depth of vortex (cm)

2.4

2.4

2.6

2.6

2.4

2.4

2.6

2.6

Observation of vortex

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

 

Cumulative mortality

Nominal loading rate (mg/L)

Number of test fish

Cumulative mortality (%)

24h

28h

72h

96h

Blank control

10

0

0

0

0

100

10

0

0

0

0

 

Observed abnormal response

Nominal loading rate (mg/L)

Result of observation

(Left column: Number affected fish / Total survival number. Right column: Symptom detail)

24h

48h

72h

96h

Blank control

0/10

N

0/10

N

0/10

N

0/10

N

100

0/10

N

0/10

N

0/10

N

0/10

N

Note: “N” means “Normal”

 

pH of test solution

Nominal loading rate (mg/L)

0h

24h

48h

72h

96h

New

Old

New

Old

New

Old

New

Old

Blank control

7.92

7.82

8.00

7.76

8.00

7.82

7.90

7.84

100

7.94

7.80

7.96

7.80

7.98

7.74

7.94

7.76

 

Dissolved oxygen concentration of test solution (%)

Nominal loading rate (mg/L)

0h

24h

48h

72h

96h

New

Old

New

Old

New

Old

New

Old

Blank control

96

92

95

89

95

90

94

89

100

95

91

96

88

93

88

96

88

 

The body weight and total length of test fish in blank control (Mean ± SD)

Index

NO. of test fish

Mean

SD

1

2

3

4

5

6

7

8

9

10

Weight (g)

0.180

0.123

0.167

0.190

0.177

0.185

0.161

0.136

0.183

0.182

0.168

0.023

Length (mm)

27.16

23.91

27.94

28.45

27.21

28.44

28.80

25.79

28.09

27.76

27.36

1.49

 

Measured concentrations of test substance in test solutions

Nominal loading rate (mg/L)

Measured concentration of test substance (mg/L)

0h (New)

24h (Old)

72h (New)

96h (Old)

Blank control

ND

ND

-

-

100

ND

ND

ND

ND

Note:     ① “-“ means not detected.

② “ND” means the measured concentration is lower than the limit of detection (LOD). The LOD is 0.863 mg/L.

 

Median Lethal Loading (LL50)

Exposure duration

LL50

24 h

>100 mg/L nominal loading rate WAF

48 h

>100 mg/L nominal loading rate WAF

72 h

>100 mg/L nominal loading rate WAF

96 h

>100 mg/L nominal loading rate WAF

 

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the test, the 96h-LL50 of test substance to Zebra fish (Danio rerio) was greater than 100mg/L nominal loading rate WAF. The highest concentration causing 0% mortality at 96h (LL0) was 100mg/L nominal loading rate WAF, the lowest concentration causing 100% mortality at 96h (LL100) was greater than 100mg/L nominal loading rate WAF.
Executive summary:

The fish are exposed to the test substance preferably for a test period of 96 hours. Mortalities and abnormalities are recorded at 24h, 48h, 72h and 96h and the median lethal concentration, i.e. the calculated concentrations of test item which causes death in 50% of the test fish (24h-LC50, 48h-LC50, 72h-LC50, 96h-LC50) are determined. At the same time, the highest test concentration without dead test organisms (LC0) and the lowest test concentration at which all test organisms are dead (LC100) within the period of the test, are determined.

 

Guidelines

[1] Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals, effects on Biotic Systems, 203 Fish Acute toxicity test [M]. China Environmental Science Press. 2013: 30~36.

[2] China National Technical Committee for Standardization of Dangerous Chemicals Management. GB/T 27861-2011, Chemicals Fish Acute toxicity test [S]. BeijingChina Standards Press, 2012.

[3] OECD. OECD Guidelines for the Testing of Chemicals, 203 Fish Acute Toxicity Test [S]. Paris: OECD, Adopted 17 July 1992.

[4] State Environmental Protection Administration of China. HJ/T 153-2004. The Guidelines for the test of chemical [S]. Beijing: China Environmental Science Press.2004.

[5] OECD. OECD series on testing and assessment number 23, Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Paris: OECD, Adopted September 2000.

[6] Standard Practice for Aquatic Toxicity Testing of Lubricants: Sample Preparation and Results Interpretation. Designation: D6081 -98 (Reapproved 2009).

[7] Standard Terminology Relating to Biodegradability and Ecotoxicity of Lubricants. Designation: D6384 - 11.

 

Test conditions

Test substance: NovaSpec Base Oil

Test organism: Zebra fish (Danio rerio)

Exposure duration: 96h

Test concentration: According to the results of other tests, a nominal loading rate of 100mg/L WAF and a blank control containing no test substance was selected.

Replicate: No replicates

Number of fish: 10 fish / test group, 20 fish in total

Test water: Dechlorinated tap water

Type of test: Semi-static test (renewal at every 24h)

Preparation of test solution: An amount of test substance (0.500g) was obtained in a flask with a small amount of test water then transferred into 5L-jar and diluted with test water to 5000mL. And the test suspension was stirred with a magnetic stirrer for about 24h, (the depth of vortex was maintained in the range of 2cm-3cm), then the nominal loading rate of 100 mg/L suspension was obtained. After the stirring was stopped, the mixture was allowed to stand for about 1 hour. Then the aqueous phase form the middle and bottom layer was removed by siphoning into another 5L-jar (the first approximated 100-200mL discarded) to give the nominal loading rate of 100 mg/L WAF (Water-accommodated fraction).

Volume of test solution: 4L / test vessel

Temperature of test environment: 22.3°C - 23.9°C

Photoperiod: 14h light / 10h darkness

Feeding: No feeding

Aeration: No aeration

Analysis of the actual concentration of test substance: The actual concentrations of 100 mg/L loading rate WAF were measured at 0h, 72h (new solution) and 24h, 96h (old solution) by GC. The blank control was measured at 0h (new solution) and 24h (old solution).

 

Results

The measured concentrations of test substance: The measured concentrations of test substance in blank control were lower than the limit of detection (LOD = 0.863 mg/L) at 0h in the new solution and at 24h in the old solution after the start of the test.

The measured concentrations of test substance in 100mg/L loading rate WAF were lower than the limit of detection (LOD = 0.863 mg/L) at 0h, 72h (new solution) and 24h, 96h (old solution) after the start of the test.

96h Median Lethal loading rate (LL50): Greater than 100mg/L nominal loading rate WAF

The highest loading rate causing 0% mortality at 96h (LL0): 100mg/L nominal loading rate WAF

The lowest loading rate causing 100% mortality at 96h (LL100): Greater than 100mg/L nominal loading rate WAF

Note: LL = Lethal Loading rate.

 

Discussion

Due to the low aqueous solubility and complex nature of test substance, so according to the guidelines of “OECD series on testing and assessment number 23, Guidance Document on Aquatic Toxic Testing of Difficult Substances and Mixtures”, the test substance solution was prepared as a Water Accommodated Fraction (WAF). During the test, the chemical analysis results showed that the measured concentration of 100mg/L loading rate WAF was lower than the limit of detection of 0.863mg/L, however, the test substance is a complex mixture, its toxicity cannot be attributed to a single component or a mixture of components, but to the test substance as a whole, the test results were based on nominal loading rate only.

Description of key information

Assessment of acute toxicity to fish

Key value for chemical safety assessment

LC50 for freshwater fish:
100 mg/L

Additional information

The substance is a hydrocarbon UVCB of limited solubility.It is unlikely that a study result would give anything other than a “greater than” limit value of the highest value available in the test. This endpoint is therefore characterized using quantitative structure property relationships for representative hydrocarbon structures that could be present within this UVCB substance. 70 proposed molecules have been assessed, in order to provide a suitable range of likely values associated with the substance.

These results, upon first inspection, indicate that the classification as “toxic” could apply. However, if the log Kow is greater than 5.0, or if the LC50 exceeds the water solubility by 10X, no effects at saturation are predicted.

Two K1 GLP studies were conducted in accoradance with OECD guidelines to assess the affects of acute toxicity to zebra fish (Danio rerio and Brachydanio rerio). The acute toxicity of the test item to fresh water Zebrafish has been investigated and gave a 96hr LC50/LL50 > 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF. The substance did not demonstrate any significant toxicity to aquatic organisms, nor does it contain significant impurities that could effect the outcome of a specific study.

Given that the substance is insoluble in water, and taken in conjunction with the acute toxicity to Danio rerio and Brachydanio rerio study results, it is clear that the substance does not demonstrate any acute toxicity effects at the limit of solubility within water. This is based on the fact that this category of substances does not demonstrate any significant toxicity to aquatic organisms, nor does it contain significant impurities that could effect the outcome of the a specific study. On this basis, it is proposed that the substance is not harmful to fish at the limit of solubility within water.

No classification and labelling is proposed.