Octadecene, reaction products with hexadecene, hydrogenated

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 29, 2019 to January 20, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

OECD Guidelines for the Testing of Chemicals. 211 . Daphnia magna Reproduction Test. Paris: OECD, Adopted 2 October, 2012.

Deviations:

yes

Remarks:

See "Any other information" for details

Qualifier:

according to guideline

Guideline:

other: National Guideline

Version / remarks:

Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals, effects on Biotic Systems, 211 Daphnia magna Reproduction Test. Second edition. Beijing: China Environmental Press. 2013: 94 - 112.

Deviations:

yes

Remarks:

See "Any other information" for details

Qualifier:

according to guideline

Guideline:

other: National Guideline

Version / remarks:

China National Technical Committee for Standardization of Dangerous Chemicals Management. GB/T 21828 - 2008, Chemicals Daphnia magna Reproduction Test. Beijing: China Standards Press, 2008.

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes

Specific details on test material used for the study:

No futher details specified in the study report.

Analytical monitoring:

yes

Details on sampling:

The test solutions were collected and filtered through 0.45 μm aqueous phase PES filter membranes (the first 5 mL - 10 mL filtrates were discarded), 50 mL filtrate was transferred to separatory funnel and extract three times with 20 mL, 15 mL and 10 mL of n-hexane, the extract solution was collected into 50 mL volume flask and diluted to 50 mL with n-hexane before used to analyze. The samples were analyzed immediately, otherwise the samples were stored at 2 °C - 8 °C and protect from light after pretreatment, and the measurement were carried out within 96 h.

Vehicle:

no

Details on test solutions:

The test substance is a UVCB substance, and it is insoluble in test water, water accommodate fraction solution was prepared as test solution. The preparation method is showed as below:
20 mg test substance was weighted into a flask and mixed with small amount of Elendt M4 medium, then transferred to 2 L beaker and diluted with Elendt M4 medium to 2000 mL, the nominal loading rate of 10 mg/L suspension was obtained. The suspension was stirred with magnetic stirrer for about 24 h (the depth of vortex was maintained at one third of the test solution height) and stand for about I h, then 1000 mL of middle layer solution was siphoned into another beaker ( discard the first I 00 mL ), the nominal loading rate of 10 mg/L Water Accommodate Fraction (WAF) solution was obtained and used as test solution.
Microscopic inspection of the WAF solution was conducted to checked micro-dispersions or un-dissolved test substance is present or not once a week.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna
Reason for selection of species: The test species is recommended by "The Guidelines for the Testing of Chemicals, effects on Biotic Systems, 211 Daphnia magna Reproduction Test".
Supplier: The parent daphnids were supplied by Safety Evaluation Center, Shenyang Research Institute of Chemical Industry. Its appearance is nominal and it bred in our laboratory. The test daphnid batch number was DMU20191206-1.
Acclimation: At the start of the test, Daphnia magna were acclimated in the testing laboratory for one generation at least under the same conditions of water quality (Elendt M4 medium), temperature (20 °C ± 2 °C), light and dark period (16 h light / 8 h dark) and illumination intensity ( 1000 Ix -1500 Ix). During the acclimation, Daphnia magna were fed with a concentrated algal suspension.
Size: The daphnids were less than 24 hours old and were not first brood progeny. The female Daphnia magna were used in the test.
Number: 20
Allocation to the test groups: The daphnids were allocated at random to each test group.

Food
Species: Pseudokirchneriella subcapitata
Supplier: Committee on Type Culture Collection of Chinese Academy of Sciences.
Condition of culture: Temperature (20 °C ± 2 °C), 24 h continuously light, illumination intensity (4440 lx-8880 Ix).

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Remarks on exposure duration:

In accordance with test guidelines

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

hardness was 245 mg/L - 260 mg/L (as CaCO3).

Test temperature:

Water temperature: 19.5 °C-20.8 °C, the total variability was < 2°C.

pH:

The pH of the test medium ranged from 7.99 to 8.47, and the vary range was less than 1.5 units during the test.

Dissolved oxygen:

The dissolved oxygen concentration in the test solutions during the test ranged from 8.4 mg/L to 9.9 mg/L, it was kept above 3 mg/L.

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

The actual concentrations of test substance in I O mg/L WAF test group were measured on 0 d, 7 d, 14 d (new solution) and 1 d, 8 d, 15 d ( old solution) by GC; the blank control were measured on 0 d (new solution) and 1 d (old solution).

Details on test conditions:

Design of the limit test
Under this test conditions, the measured concentrations of test substance in test solution were all lower than limit of detection, consequently the limit test was perfonned under semi-static test at 24 h intervals. According to results of preliminary tests, one blank control and one nominal loading rate of 10 mg/L water accommodate fraction test group were set in the limit test. 10 replicate per test group, 1 parent animal for each replicate (vessel).

Preparation of the test system
(1) Blank control: Elendt M4 test medium was divided into 10 test vessels, respectively, and the volume of test medium was 80 mL for each test vessel as the blank control group. Another parallel vessel was also prepared for the measurement of pH, temperature, dissolved oxygen (DO) and the total hardness at the start of the test and before renewal solution.
(2) Test substance group: 10 mg/L WAF test solution was divided into 10 test vessels, respectively, and the volume of test medium was 80 mL for each test vessel as the test substance group. Another parallel vessel was also prepared for the measurement of pH, temperature, DO and total hardness at the start of the test and before renewal solution.

Test conditions
Conditions of exposure
(1) Type of test: Semi-static test (renewal at every 24 h)
(2) Exposure duration: 21 d
(3) Test concentration: The test was conducted with a nominal loading rate of 10 mg/L WAF test group.
(4) Control: A control (test medium without test substance) was run in parallel to the test concentrations.
(5) Replicate: 10 replicates
(6) Number of daphnid: 10 / test group, 20 in total
(7) Volume of test solution: 80 mL / test vessel

Conditions of test environment
(1) Water temperature: 19.5 °C-20.8 °C, the total variability was < 2°C.
(2) Dissolved oxygen concentration: The test solutions were not aerated during the exposure. The dissolved oxygen concentration in the test solutions during the test ranged from 8.4 mg/L to 9.9 mg/L, it was kept above 3 mg/L.
(3) pH: The test was perfonned without adjustment of pH. The pH of the test medium ranged from 7.99 to 8.47, and the vary range was less than 1.5 units during the test.
(4) Light: Artificial light of white fluorescent lamp, 16 h light / 8 h darkness, the average illumination intensity was 1291 lx and 1319 lx at the start and at the end of the test.
(5) Feeding: The daphnids were fed with a concentrated algal suspension daily during the exposure. The diet of the parent daphnids was about 0.1 mg C/Daphnia/day - 0.2 mg C/Daphnia/day.

Observation and measurements
(1) Observation, mortality and body length of parent daphnias
The appearance and mortality of parent daphnias were recorded daily. At the end of the test, the body length of each surviving parent daphnias (anal spine not included) was detennined using digital caliper (The precision was 0.01 mm).

(2) The number oflive offspring
The live offspring produced by each parent daphnid was removed and counted daily, and also the presence of dead offspring and aborted eggs were recorded.

(3) Measurement of intensity of illumination and temperature, pH, dissolved oxygen, total hardness of test solution
The intensity of illumination of test environment was measured at the start and the end of the test, respectively.
The water temperature, pH, DO and hardness of blank control and 10 mg/L WAF test group were measured on O d, 7 d and 14 d in new solutions and on 1 d, 8 d and 15 d in old solutions. The temperature of the test solution was continuously monitored during the test exposure.

Data processing
The average and standard deviation of survival offspring and the body length of parent daphnids in each test group were calculated at the end of test. The different of number of offspring and body length of parent daphnids in blank control and test group were compared by using "Independent sample T-test". The results of comparison were used to estimate the LOEL and NOEL.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

10 other: mg/L (WAF)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

10 other: mg/L (WAF)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

10 other: mg/L (WAF)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Key result

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

>= 10 other: mg/L (WAF)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

>= 10 other: mg/L (WAF)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

>= 10 other: mg/L (WAF)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Details on results:

Observation of test solutions
The WAF solution was prepared with the direct addition method. During the preparation periods, surface films in the top layers were observed in all test solutions, so the solution in the middle layer was siphoned into another test vessel as exposure solution, certainly, there no obvious droplets or surface films were observed in the exposure solution. Any remaining non-dissolved test chemical component in the WAF has the possibility to cause physical effects on the test organism unrelated to inherent chemical toxicity.

Observations of parent daphnids
During test period, there was no abnonnal color and responses of parent daphnids in blank control and the 10 mg/L WAF test group.
The time to the presence of the first eggs in the brood pouch in blank control and 10 mg/L WAF test group were 5.6 ± 1.0 d and 5.8 ± 1.0 d respectively, and the time to production of first brood in blank control and 10 mg/L WAF test group were 8.6 ± 1.0 d and 8.7 ± 1.0 d respectively.

Mortality rate of parent daphnids
At the end of the test, there were no parent daphnids dead in the blank control group, which met the validity criteria that the mortality of the parent daphnids should not exceed 20% at the end of the test.
At the end of the test, the mortality of parent daphnids in the 10 mg/L WAF test group was 0%. So according to the mortality of parent daphnid, LOEL was greater than the nominal loading rate of 10 mg/L (WAF), NOEL was the nominal loading rate of 10 mg/L (WAF).

Body length of the parent daphnids
Results of Independent samples T-test shown that, there was no significant different (p>0.05) on body length of parent animals between the blank control and 10 mg/L WAF test group. So according to the body length of the parent daphnids, LOEL was greater than the nominal loading rate of 10 mg/L (WAF), NOEL was the nominal loading rate of 10 mg/L (WAF).

Living offspring by each parent daphnid
At the end of the test, the mean ± Standard Deviation (SD) of reproductive output of the control group was 164 ± 9 offspring per surviving parent daphnid. The coefficient of variation in the control group was 5%.
No dead offspring and aborted broods were observed in blank control and 10 mg/L WAF test group during the test.
The mean ± SD of living offspring produced per surviving parent daphnid in each test group were estimated by Independent T-test. Results showed that there was no significant different (p>0.05) on number of living offspring between the blank control and 10 mg/L WAF test group. So according to the reproductive output, LOEL was greater than the nominal loading rate of 10 mg/L (WAF), NOEL was the nominal loading rate of 10 mg/L (WAF).

Measurements of illumination and water temperature, pH, DO and hardness
Intensity values of illumination at the start and the end of the test were 1291 lx and 1319 Ix, respectively. During the test, the result of continuous monitoring of the water temperature was shown that the water temperature was 19.5 °C - 20.8 °C.
The pH, DO and hardness of blank control and 10 mg/L WAF test group were measured on 0 d, 7 d and 14 d (new solution) and 1 d, 8 d and 15 d ( old solution). During of the test, pH of test solutions was 7.99 - 8.47, DO was 8.4 mg/L - 9.9 mg/L, hardness was 245 mg/L - 260 mg/L (as CaCO3).

Measurement of test substance concentrations in test solutions
Sample pretreatment and preservation
The test solutions were collected and fi ltered through 0.45 μm aqueous phase PES filter membranes (the first 5 mL - 10 mL filtrates were discarded), 50 mL filtrate was transferred to separatory funnel and extract three times with 20 mL, 15 mL and 10 mL of n-hexane, the extract solution was collected into 50 mL volume flask and diluted to 50 mL with n-hexane before used to analyze. The samples were analyzed immediately, otherwise the samples were stored at 2 °C - 8 °C and protect from light after pretreatment, and the measurement were carried out within 96 h.

The results of the measurement of test substance concentrations
The limit of detection (LOD) of test substance analytical method was 0.645 mg/L, and the Limit of quantitation (LOQ) was 2.15 mg/L.
The measured concentrations of test substance in blank control were all lower than 0.645 mg/L (LOD) on 0 d (new solution) and 1 d (old solution) after the start of the test.
The measured concentrations of test substance in 10 mg/L W AF test group on 0 d, 7 d, 14 d (new solution) and 1 d, 8 d, 15 d ( old solution) were all lower than 0.645 mg/L (LOD). The measured concentrations of all test solutions lower than the LOD, this did not mean that there was no test substance presented in the test solutions, just that the concentration was below the LOD of the analytical method, indicating that test substance was poorly soluble in test water.

Results with reference substance (positive control):

Reference substance not utilised for the study.

Reported statistics and error estimates:

(I) The daphnids were less than 24 hours old and were first brood progeny.
(2) The mortality of the parent daphnids (female Daphnia) was 0%, which was not exceed 20% at the end of the test in the control.
(3) The mean number of live offspring produced per parent daphnid surviving was 164 at the end of the test in the control, which was greater than 60.
(4) The coefficient of variation around the mean number of living offspring produced per parent daphnid in the control was 5%, which was lower than 25%,

Observations of parent daphnids during test period

Nominal loading rate

Replicate

Observation of parent daphnids

0d

1d

2d

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10d

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Blank control

1

AN

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10 mg/L WAF

1

AN

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4

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AN+

Note: “AN” means appears normal, “P” means onset of production. “+” means eggs present in brood pouch.

 

The mean ± SD of pregnancy and first reproduction

Nominal loading rate	Mean ± SD of first pregnancy (d)	Mean ± SD of first reproduction (d)
Blank control	5.6 ± 1.0	8.6 ± 1.0
10 mg/l WAF	5.8 ± 1.0	8.7 ± 1.0

 

The mortality of parent daphnid per group at the end of the test

Nominal loading rate	Total	Mortality	Mortality rate (%)
Blank control	10	0	0
10 mg/L WAF	10	0	0

 

The mean ± SD of body length of parent daphnid alive at the end of the test

Nominal loading rate	Mean ± SD of body length (mm)
Blank control	4.04 ± 0.06
10 mg/L WAF	4.01 ± 0.11

Note: SD = Standard deviation

 

The total number of living offspring produced per parent daphnid during the test

Nominal loading rate	Replicate	The number of living offspring produced per parent daphnid
		8d	9d	10d	11d	12d	13d	14d	15d	16d	17d	18d	19d	20d	21d	Total number
Blank control	1	11	0	0	30	0	0	46	0	0	0	28	0	0	47	162
	2	6	0	0	37	0	0	41	0	0	31	0	0	0	51	166
	3	12	0	0	0	21	0	0	37	0	0	29	0	0	37	146
	4	16	0	0	35	3	0	37	0	0	0	33	0	0	45	169
	5	14	0	0	15	18	0	34	0	0	0	35	0	0	40	156
	6	15	0	0	0	36	0	40	0	0	0	38	0	0	47	176
	7	0	0	37	0	0	0	0	0	47	0	0	30	4	0	161
	8	11	0	0	34	0	43	46	0	0	0	34	0	0	46	171
	9	0	0	34	0	0	0	0	0	53	0	0	40	2	0	166
	10	0	0	34	0	0	37	0	0	51	0	0	1	37	0	166
10 mg/L WAF	1	10	0	0	35	0	43	45	0	0	0	41	0	0	52	183
	2	11	0	0	34	0	0	48	0	0	42	0	0	60	0	195
	3	17	0	0	36	0	0	31	0	0	0	35	0	0	42	161
	4	12	0	0	1	35	0	31	0	0	0	30	0	0	45	154
	5	17	0	0	36	0	0	40	0	0	0	46	0	0	56	195
	6	0	0	30	0	0	40	0	0	0	38	0	0	48	0	156
	7	0	0	34	0	0	41	0	0	0	40	0	0	47	0	162
	8	0	0	28	0	0	38	0	0	45	0	0	38	0	0	149
	9	0	10	0	0	26	0	0	29	0	0	32	0	0	44	141
	10	14	0	0	1	26	0	36	0	0	0	39	0	0	50	176

 

The mean ± SD of living offspring produced per daphnid alive at the end of the test

Nominal loading rate	Mean ± SD
Blank control	164 ± 9
10 mg/L WAF	167 ± 20

Note: SD = Standard deviation

 

Measured intensity of illumination at the start and the end of the test

Time	Intensity of illumination (lx)
	1	2	3	Mean value
The start	1150	1310	1412	1291
The end	1226	1380	1352	1319

 

Water pH, DO and hardness (as CaCO3) in test solutions

Nominal loading rate	Test indicators	Results
		0 d new	1 d old	7 d new	8d old	14 d new	15 d old
Blank control	pH	8.31	8.47	8.28	8.16	8.26	8.05
	DO (mg/L)	8.5	9.9	8.6	9.5	8.8	9.0
	Hardness (mg/L)	245	245	250	250	250	260
10 mg/L WAF	pH	8.20	8.34	8.15	8.11	8.10	7.99
	DO (mg/L)	8.4	9.6	8.5	9.3	8.9	9.0
	Hardness (mg/L)	245	245	250	250	255	260

 

The measured concentrations of test substance in the test solution

Nominal loading rate	Measured concentration (mg/L)
	0 d new	1 d old	7 d new	8 d old	14 d new	15 d old
Blank control	<LOD	<LOD	-	-	-	-
10 mg/L WAF	<LOD	<LOD	<LOD	<LOD	<LOD	<LOD

Note: the limit of detection (LOD) and limit of quantification (LOQ) of test substance were 0.645 mg/L and 2.15 mg/L respectively, “<LOD” means lower than LOD, “-“ means have no detection.

Validity criteria fulfilled:

yes

Conclusions:

In this test, all the validity criteria were met with requirements. The results of LOEL and the NOEL are shown as follow:
21d Lowest Observed Effect Loading rate (LOEL)
LOEL for effects on reproduction > nominal loading rate of 10 mg/L (WAF)
LOEL for effects on body length > nominal loading rate of 10 mg/L (WAF)
LOEL for mortality of parent daphnids > nominal loading rate of 10 mg/L (WAF)

21d No Observed Effect Loading rate (NOEL)
NOEL for effects on reproduction = nominal loading rate of 10 mg/L (WAF)
NOEL for effects on body length = nominal loading rate of 10 mg/L (WAF)
NOEL for mortality of parent daphnids = nominal loading rate of 10 mg/L (WAF)

Executive summary:

Test substance: SynNova Base Oil

Test organism: Daphnia magna

Exposure duration: 21 days (d)

 

Test concentration: According to the results from preliminary tests, the limit test was performed with a nominal loading rate of 10 mg/L Water Accommodate Fraction (WAF) test group and a blank control.

 

Replicate: 10 replicates / test group

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

Test medium: Elendt M4 medium

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

 

Preparation of test solution: 20 mg test substance was weighted into a flask and mixed with small amount of Elendt M4 medium, then transferred to 2 L beaker and diluted with Elendt M4 medium to 2000 mL, the nominal loading rate of 10 mg/L suspension was obtained. The suspension was stirred with magnetic stirrer for about 24 h (the depth of vortex was maintained at one third of the test solution height) and stand for about 1 h, then I 000 mL of middle layer solution was siphoned into another beaker (discard the first 100 mL), the nominal loading rate of 10 mg/L WAF solution was obtained and used as test solution.

This procedure is in accordance with the OECD guidance document No. 23 (2019) andASTM D6081 (2014).

 

Preparation of test system: 10 mg/L WAF was divided into 10 test vessels, and the volume of test medium was 80 mL for each test vessel as the test substance group. Elendt M4 test medium was divided into 10 test vessels, respectively, and the volume of test medium was 80 mL for each test vessel as the blank control group. One test daphnid and feed, concentrated algal suspension, were added into test vessel to start the test.

 

Volume of test solution: 80 mL / test vessel

Temperature of test solutions: 19.5 °C -20.8 °C

Photoperiod: 16 h illumination / 8 h darkness.

Feeding: A concentrated algal suspension was fed to the Daphnia daily, the supplied diet was 0.1 mg C/Daphnid/day - 0.2 mg C/Daphnia/day.

Aeration: No aeration

 

Analysis of the actual concentrations of test substance in test solutions: The actual concentrations of test substance in 10 mg/L WAF test group were measured on 0 d, 7 d, 14 d (new solution) and 1 d, 8 d, 15 d (old solution) by Gas Chromatograph (GC); the blank control were measured on 0 d (new solution) and 1 d (old solution).

 

Results

Basis of preparation test solution

The SynNova Base Oil is Liquid - water white colorless oil. The test substance is a complex mixture / UVCB substance (Substances of Unknown or Variable Composition, Complex Reaction Products or Biological Materials) and is poorly soluble in test water. The aqueous media were prepared following ASTM D6081 -98/Reapproved 2014), which is specifically developed for aquatic toxicity testing of lubricants, by mixing the test substance with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. At the completion of mixing and following a settlement period, the WAF was separated by siphon. The WAF was checked for any undissolved or emulsified material by microscopic inspection, which was negative. Exposure was expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate). This procedure was followed for each renewal of the test solutions.

 

The measured concentrations of test substance in test solutions

The limit of detection (LOD) of test substance analytical method was 0.645 mg/L, and the Limit of quantitation (LOQ) was 2.15 mg/L.

The measured concentrations of test substance in blank control were all lower than 0.645 mg/L (LOD) on 0 d (new solution) and 1 d (old solution) after the start of the test.

The measured concentrations of test substance in 10 mg/L W AF test group on 0 d, 7 d, 14 d (new solution) and 1 d, 8 d, 15 d (old solution) were all lower than 0.645 mg/L (LOD). The measured concentrations of all test solutions lower than the LOD, this did not mean that there was no test substance presented in the test solutions, just that the concentration was below the LOD of the analytical method, indicating that test substance was poorly soluble in test water.

 

Toxicity effect of test organism

In this test, all the validity criteria were met with requirements. The results of LOEL and the NOEL are shown as follow:

 

21 d Lowest Observed Effect Loading rate (LOEL)

LOEL for effects on reproduction > nominal loading rate of 10 mg/L (WAF)

LOEL for effects on body length > nominal loading rate of 10 mg/L (WAF)

LOEL for mortality of parent daphnids > nominal loading rate of 10 mg/L (WAF)

 

21d No Observed Effect Loading rate (NOEL)

NOEL for effects on reproduction = nominal loading rate of 10 mg/L (WAF)

NOEL for effects on body length = nominal loading rate of 10 mg/L (WAF)

NOEL for mortality of parent daphnids = nominal loading rate of 10 mg/L (WAF)

Description of key information

21 d Lowest Observed Effect Loading rate (LOEL)

LOEL for effects on reproduction/bdoy length/mortality of parent daphnids > nominal loading rate of 10 mg/L (WAF)

 

21d No Observed Effect Loading rate (NOEL)

NOEL for effects on reproduction/body length/mortality of parent daphnids = nominal loading rate of 10 mg/L (WAF)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

10 mg/L

Additional information

The SynNova Base Oil is Liquid - water white colorless oil. The test substance is a complex mixture / UVCB substance (Substances of Unknown or Variable Composition, Complex Reaction Products or Biological Materials) and is poorly soluble in test water. The aqueous media were prepared following ASTM D6081 -98/Reapproved 2014), which is specifically developed for aquatic toxicity testing of lubricants, by mixing the test substance with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. At the completion of mixing and following a settlement period, the WAF was separated by siphon. The WAF was checked for any undissolved or emulsified material by microscopic inspection, which was negative. Exposure was expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate). This procedure was followed for each renewal of the test solutions.

 

The limit of detection (LOD) of test substance analytical method was 0.645 mg/L, and the Limit of quantitation (LOQ) was 2.15 mg/L.

The measured concentrations of test substance in blank control were all lower than 0.645 mg/L (LOD) on 0 d (new solution) and 1 d (old solution) after the start of the test.

The measured concentrations of test substance in 10 mg/L W AF test group on 0 d, 7 d, 14 d (new solution) and 1 d, 8 d, 15 d (old solution) were all lower than 0.645 mg/L (LOD). The measured concentrations of all test solutions lower than the LOD, this did not mean that there was no test substance presented in the test solutions, just that the concentration was below the LOD of the analytical method, indicating that test substance was poorly soluble in test water.

 

Toxicity effect of test organism

In this test, all the validity criteria were met with requirements. The results of LOEL and the NOEL are shown as follow:

 

21 d Lowest Observed Effect Loading rate (LOEL)

LOEL for effects on reproduction > nominal loading rate of 10 mg/L (WAF)

LOEL for effects on body length > nominal loading rate of 10 mg/L (WAF)

LOEL for mortality of parent daphnids > nominal loading rate of 10 mg/L (WAF)

 

21d No Observed Effect Loading rate (NOEL)

NOEL for effects on reproduction = nominal loading rate of 10 mg/L (WAF)

NOEL for effects on body length = nominal loading rate of 10 mg/L (WAF)

NOEL for mortality of parent daphnids = nominal loading rate of 10 mg/L (WAF)