Registration Dossier

Ecotoxicological information

Long-term toxicity to fish

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
adult fish: sub(lethal) effects
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 4, 2014 to December 23 , 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed to national guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
other: Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals, Effects on Biotic Systems, 204 Fish, Prolonged Toxicity Test: 14 day Study [M]. Second Edition. Beijing: China Environmental Press. 2013:37-41.
Deviations:
no
Qualifier:
according to
Guideline:
other: China National Technical Committee for Standardization of Dangerous Chemicals Management. GBrr 2 1808 - 2008 Chemicals-Fish, Prolonged Toxicity: 14-Day Study Test [S]. Beijing: Standards Press of China, 2008.
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD. OECD series on testing and assessment number 23, Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures [S]. Paris: OECD, Adopted September 2000.
Deviations:
no
Qualifier:
according to
Guideline:
other: Standard Practice for Aquatic Toxicity Testing of Lubricants: Sample Preparation and Results Interpretation. Designation: D6081-98 (Reapproved 2009).
Deviations:
no
Qualifier:
according to
Guideline:
other: State Environmental Protection Administration of China. HJff 153-2004 The Guidelines for the test of chemical [S] . Beijing: China Environmental Science Press. 2004.
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):None specified
Analytical monitoring:
yes
Details on sampling:
The actual concentrations of 100 mg/L nominal loading rate WAF were measured with GC at 0d, 5d, 9d, 13d in the new solution and at 1d, 6d, 10d, 14d in the old solution. The blank control was measured at 0d (new solution) and 1d (old solution).
Vehicle:
no
Details on test solutions:
An amount of test substance (0.500 g - 0.504 g) was obtained in a flask with a small amount of test water then transferred into 5L-jar and diluted with test water to 5000 mL. And the test solution was stirred with a magnetic stirrer for about 24h, (the depth of vortex was maintained in the range of 2 cm – 3 cm) 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 from the middle and bottom layer was removed by siphoning into another 5L-jar (the first approximated 100-200 mL discarded) to give the nominal loading rate of 100 mg/L WAF (water-accommodated fraction) solution.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Species: Zebrafish (Danio rerio)Reason for selection of species: The test species is recommended by "The Guidelines for the Testing of Chemicals".Supplier: Guangzhou Yangshi Breeding FarmAcclimationZebra fish (Danio rerio) were obtained on October 23, 2014. The Batch number wasFDr20 141023-1.Before the start of the test, Zebra fish (Danio rerio) were acclimated for 43 days under the same conditions (water quality: dechlorinated tap water; temperature: 23 °C ± 2 °C; photoperiod: 14 h light / 10 h 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.The mortality was 1.2% 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 2014GST0016) showed that the 24h-LC50 of the reference substance (K2Cr2O7) was 252mg/L to Zebra fish (Danio rerio) and the 95% confidence limits were 223 mg/L to 284 mg/L. The results were kept at the value ranging from 200 mg/L to 400 mg/L, which met the criterion for the validity of the test.Total Number of test fish30 fish were used in the whole test (the sex of fish was not determined). 10 representative fish of them were used for the measurement of body weight and body length before the start of the test.Allocation to the test groupsZebra fish (Danio rerio) were randomly picked out and allocated at random to each test group within 30 minutes.FodderName: Frozen red wormIngredient: Crude protein 8.5%, crude fat 1.2%, crude fibre 0.8%, moisture 88%
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
14 d
Post exposure observation period:
No post exposure observation period specified in the study report.
Hardness:
118mg!L (expressed as CaC03)
Test temperature:
The temperature of test environment water ranged from 21.0°C to 23.9°C.
pH:
pH ranged from 7.76 to 8.29
Dissolved oxygen:
dissolved oxygen ranged 80% to 101%
Salinity:
Not specified
Nominal and measured concentrations:
The actual concentrations of I 00 mg/L nominal loading rate WAF were measured with GC at 0d, 5d, 9d, 13d in the new solution and at 1d, 6d, 10d, 14d in the old solution. The blank control was measured at 0d (new solution) and 1d (old solution).
Details on test conditions:
Conditions of exposure
Type of test:
The measured concentrations of the test substance in the solution were all lower than the limit of detection (LOD = 0.863 mg/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: 14 days
Test concentration: According to the results of other tests, a nominal loading rate of 100mg/L WAF test group was selected.
Control: A blank control (test water without test substance) was also run in parallel to the test concentration.
Replicate: No replicates / test group
Number of fish: 10 fish / test group
Volume of test solution: 4L / test vessel
Loading: 10 fish / test vessel, 0.359 g fish / L
Conditions of test environment
Water temperature: 21.0°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. The range of pH was 7.76 to 8.29
.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 89 lx and 82 lx on average, respectively.
Feeding: Feed with red worm ad libitum once a day. The ration of worm was about 200 mg, equal to 1%-2% of the total body weight of fish per test group.
Remove contamination: Clean the vessel when renewing the test solution.
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 were also recorded.
Observations on test substance solubility: At the start and end of each mixing period, and after the 1-Hour standing period the appearance of the WAF solution was observed and recorded. After siphoning, the WAF solution was inspected under microscope.
Measurement of light illumination: The intensity of illumination was measured at the beginning and end of the test.
Observation of test organisms: The mortality of the test fish was recorded every day. A fish was considered dead if there was no visible movement (e.g. gill movements) and if touching of the caudal peduncle produced no reaction. The dead fish were removed as soon as they were found.The visible abnormality was recorded on 1d, 3d, 5d, 7d, 9d, 11d, 13d and 14d after the start of the test during test period. The Non-lethal effects of test fish including the different appearance, size, behavior, such as abnormal swimming, different reaction to external stimulus, different appearance, reduction or cease of feeding, change of body weight or body length, comparing with blank control. The changes of color and appearance of the test suspension were recorded at the same time.Measurement of temperature, pH and dissolved oxygen of test solution: The temperature, pH and dissolved oxygen of each test group were measured at 0d, 5d, 9d, and 13d in the new solutions and measured at 1d, 6d, 10d and 14d in the old solutions after the start of the test. And the temperature of test water was continuously monitored during the test exposure by dual thermometer.Body weight and body length: The body weight and body length of 10 representative fish before the test were determined, which was used for calculating the feeding quantity and loading during the test period. And all test fish alive at the end of the test were also measured.Measurement of actual concentrations of test substance: The actual concentrations of I 00 mg/L nominal loading rate WAF were measured with GC at 0d, 5d, 9d, 13d in the new solution and at 1d, 6d, 10d, 14d in the old solution. The blank control was measured at Od (new solution) and 1d (old solution).
Reference substance (positive control):
no
Duration:
14 d
Dose descriptor:
other: LOEL
Effect conc.:
> 100 other: mg/L nominal loading rate WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
14 d
Dose descriptor:
other: NOEL
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 solubilityAt the start and end of each mixing period, the 100mg/L loading rate 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 100 mg/L loading rate WAF solution was observed to remain a clear colorless water column in the mid-depth but with oily globules of test substance floating on the water surface. After siphoning, the microscopic inspection of WAF showed no micro-dispersions or undissolved test substance to be present.

Observation of test solutions
The blank control and the 100 mg/L loading rate WAF test solutions were observed to be clear and colorless during the test period.Intensity of illuminationAt the start of the test, the intensity of illumination of test environment was 70 lx, 90 lx and 106 lx (89 lx on average).At the end of the test, the intensity of illumination of test environment was 65 lx, 84 lx and 96 lx (82 lx on average).

Mortality
The cumulative mortality of blank control was 0% at the end of the test, which met the criterion for the validity of the test. At the end of the test, the cumulative mortality of the nominal loading rate of 100 mg/L WAF was 0%. So the 14 day threshold level of lethal effect of test substance was greater than 100 mg/L nominal loading rate WAF.

Observed abnormal response
At the end of the test, no abnormal responses were observed in blank control and the test substance group. So the 14 d lowest observed effect loading rate (LOEL) was greater than 100 mg/L nominal loading rate WAF. The 14d no observed effect loading rate (NOEL) was 100 mg/L nominal loading rate WAF.

Body weight and body length
The body weight and body length of 10 representative fish before the test were 0.144 g ± 0.016 g and 23.79 mm ± 0.99 mm respectively.The body weight and body length of all fish alive in blank control and the test substance group at the end of the test were 0. 146 g ± 0.017 g, 0.153 g ± 0.016 g and 24.48 mm ± 1.31 mm, 24.19 mm ± 1.65 mm, respectively.

Measurement of temperature, pH and dissolved oxygen of test solution
During the test, the measured values of pH and dissolved oxygen ranged from 7.76 to 8.29, and 80% to 101%, respectively. The temperature of test environment water ranged from 21.0 °C to 23.9 °C. The dissolved oxygen concentrations were kept at least 60 percent of the air saturation value, which met the criterion for the validity of the test.Determination of test substance concentration

Sample pre-treatment and preservation
50 mL test solutions were accurately taken and poured into the separating funnel, then 20 mL, 15 mL, 10 mL hexyl hydride were added respectively to extract for three times (5 min for each time). After that, extractions were collected and transferred into a 50 mL volumetric flask and then diluted with hexyl hydride to 50 mL. 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 of test substance
The results of the analytical method are given in the final report of "Validation of an Analytical Method of NovaSpec Base Oil" (The report No. is A 14048-T14037-1R).The measured concentrations of test substance in blank control were lower than the limit of detection (LOD = 0.863 mg/L) at 0d in the new solution and at Id in the old test solution after the start of the test.The measured concentrations of test substance in 100 mg/L loading rate WAF test group were lower than the limit of detection (LOD = 0.863 mg/L) at 0d, 5d, 9d, 13d in the new solution and at 1d, 6d, 10d, 14d in the old solution.
Results with reference substance (positive control):
Positive control not performed in this study.
Reported statistics and error estimates:
The mortality of the control group should not be more than 10% at the end of the test.The concentration of the dissolved oxygen in the solution should be kept at least 60 percent of the air saturation value throughout the test.

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

100 mg/L loading rate WAF test group

Measurements

0d

1d

2d

3d

4d

5d

6d

Start

End

Start

End

Start

End

Start

End

Start

End

Start

End

Start

End

Depth of vortex (cm)

2.0

2.0

2.2

2.2

2.1

2.1

2.2

2.3

2.4

2.5

2.3

2.5

2.4

2.5

Observation of vortex

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

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

100 mg/L loading rate WAF test group

Measurements

7d

8d

9d

10d

11d

12d

13d

Start

End

Start

End

Start

End

Start

End

Start

End

Start

End

Start

End

Depth of vortex (cm)

2.2

2.4

2.3

2.2

2.4

2.3

2.2

2.1

2.4

2.1

2.3

2.5

2.2

2.1

Observation of vortex

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

Dimple

 

Cumulative mortality

Nominal loading rate (mg/L)

Number of test fish

Cumulative mortality (%)

1d

2d

3d

4d

5d

6d

7d

8d

9d

10d

11d

12d

13d

14d

Blank control

10

0

0

0

0

0

0

0

0

0

0

0

0

0

0

100

10

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Observed abnormal response

Nominal loading rate (mg/L)

Result of observation

(Number of affected fish /Total survival number, Abnormality detail)

1d

3d

5d

7d

9d

11d

13d

14d

Blank control

0/10

N

0/10

N

0/10

N

0/10

N

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

0/10

N

0/10

N

0/10

N

0/10

N

Note: N-Normal

 

The body weight and body length of 10 representative fish before the test and

all fish alive at the end of the test

Time

Body weight (g)

Body length (mm)

Before the test *

0.144 g ± 0.016 g

23.79 mm ± 0.99 mm

At the end of the test

Blank control

0.146 g ± 0.017 g

24.48 mm ± 1.31 mm

100 mg/L WAF

0.153 g ± 0.016 g

24.19 mm ± 1.65 mm

Note: “*” Measurement of the body weight and body length of 10 representative fish

before the test.

 

pH of test solutions

Nominal loading rate (mg/L)

0d

(new)

1d

(old)

5d

(new)

6d

(old)

9d

(new)

10

(old)

13d

(new)

14

(old)

Blank control

7.93

7.82

7.98

7.80

7.92

7.89

7.81

7.76

100

8.29

7.92

8.24

7.76

7.96

8.02

8.13

7.92

 

Dissolved oxygen concentration of test solutions (%)

Nominal loading rate (mg/L)

0d

(new)

1d

(old)

5d

(new)

6d

(old)

9d

(new)

10

(old)

13d

(new)

14

(old)

Blank control

100

96

100

94

98

93

97

94

100

101

94

99

92

80

90

100

92

 

Measured concentrations of test substance in test solutions

Nominal loading rate (mg/L)

0d

(new)

1d

(old)

5d

(new)

6d

(old)

9d

(new)

10

(old)

13d

(new)

14

(old)

Blank control

ND

ND

-

-

-

-

-

-

100

ND

ND

ND

ND

ND

ND

ND

ND

Note: “-“ means not detected. “ND” means the measured concentration was lower than the limit of detection (LOD);

The LOD is 0.863 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the test, the 14 day threshold level of lethal effect of test substance to Zebra fish (Dania rerio) was greater than 100 mg/L nominal loading rate WAF. The 14 day lowest observed effect loading rate (LOEL) was greater than 100 mg/L nominal loading rate WAF. The 14 day no observed effect loading rate (NOEL) was 100 mg/L nominal loading rate WAF.
Executive summary:

The aim of the study is to evaluate the Prolonged Toxicity of the test substance to fish over a 14-day test period. Zebrafish (Dania rerio) were exposed to aqueous test media containing the test item under defined conditions. Recorded effects were the threshold levels of lethality and other observed effects and the NOEC for the test fish.

 

Guidelines

-Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals, Effects on Biotic Systems, 204 Fish, Prolonged Toxicity Test: 14 day Study. Second Edition. Beijing: China Environmental Press. 2013:37-41.

-China National Technical Committee for Standardization of Dangerous Chemicals Management. GB/T 21808 - 2008 Chemicals-Fish, Prolonged Toxicity: 14-Day Study Test. Beijing: Standards Press of China, 2008.

-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.

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

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

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

 

Test substance: NovaSpec Base Oil

Test organism: Zebra fish (Danio rerio)

Exposure duration: 14d

Test concentration: According to the results of other tests, a nominal loading rate of 100 mg/L water-accommodated fraction (WAF) solution test group and a blank control containing no test substance was selected.

Replicate: No replicates / test group

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 (0.500 g - 0.504 g) was obtained in a flask with a small amount of test water then transferred into 5L-jar and diluted with test water to 5000 mL. And the test solution was stirred with a magnetic stirrer for about 24h (the depth of vortex was maintained in the range of 2 cm – 3 cm), 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 from the middle and bottom layer was removed by siphoning into another 5L-jar (the first approximated 100 – 200 mL discarded) to give the nominal loading rate of 100 mg/L WAF (water-accommodated fraction) solution.

Volume of test solution: 4L / test vessel

Temperature of test solutions: 21.0 °C -23.9 °C

Photoperiod: 14h light / 10h darkness

Feeding: Fed with red worm once a day

Aeration: No aeration

Analysis of the actual concentration of test substance in test solution: The actual concentrations of 100 mg/L loading rate WAF test group were measured at 0d, 5d, 9d, 13d in the new solution and at 1d, 6d, 10d, 14d in the old solution by GC. The blank control were measured at 0d (new solution) and 1d (old solution).

 

Results

The measured concentrations of test substance in test solution: The results of the analytical methods are given in the final report of "Validation of an Analytical Method of NovaSpec Base Oil" (The report No. is A 14048-T14037-1R).

The measured concentrations of test substance in blank control were lower than the limit of detection (LOD = 0.863 mg/L) at 0d in the new solution and at 1d in the old test solution after the start of the test. The measured concentrations of test substance in 100 mg/L loading rate WAF were lower than the limit of detection (LOD = 0.863 mg/L) at 0d, 5d, 9d, 13d in the new solution and at 1d, 6d, 10d, 14d in the old solution.

 

14d threshold level of lethal effect: Greater than 100 mg/L nominal loading rate WAF

14d lowest observed effect loading rate (LOEL): Greater than 100 mg/L nominal loading rate WAF

14d no observed effect loading rate (NOEL): 100 mg/L nominal loading rate WAF

 

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 100 mg/L loading rate WAF was lower than the limit of detection of 0.863 mg/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.

No classification is applicable.

Description of key information

Assessment of chronic toxicity to fish

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
100 mg/L

Additional information

A K1 GLP study was conducted in accoradance with OECD guidelines to assess the affects of chronic toxicity to Zebrafish (Danio rerio). The chronic toxicity of the test item to fresh water Zebrafish has been investigated and gave a 14 day Lowest Observed Effect Loading rate value > 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 the a specific study.

No classification is applicable