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EC number: 939-894-0 | CAS number: -
Vortex depth and observation measurements at the start and end of mixing period
100 mg/L loading rate WAF test group
Depth of vortex (cm)
Observation of vortex
Vortex depth and observation measurements at the start and end of mixing period (continued)
The total number of living offspring produced per parent animal during the test
Nominal loading rate (mg/L)
The number of living offspring produced per parent animal
The mortality of parent animal per group at the end of the test
The mean ± SD of living offspring produced per parent animal alive at the end of the test
Mean ± SD
148 ± 20
145 ± 22
Note: SD = Standard deviation
The mean ± SD of body length of parent animal alive at the end of the test
3.97 ± 0.096
3.98 ± 0.078
Measured intensity of illumination at the start and the end of the test respectively
Intensity of illumination (lx)
pH, DO and hardness in test solutions
Nominal loading rate 100 mg/L WAF
The primary objective of the test is to assess the effect of chemicals on the reproductive output of Daphnia magna. To this end, young female Daphnia (the parent animals), aged less than 24 hours at the start of the test, are exposed to the test substance added to water at a range of concentrations. The test duration is 21 days. At the end of the test, the total number of living offspring produced is assessed. The reproductive output and growth of the adult daphnias exposed to the test substance is compared to that of the control(s) in order to determine the lowest observed effect loading rate (LOEL) and hence the no observed effect loading rate (NOEL). In addition, and as far as possible, the data are analysed using a regression model in order to estimate the loading rate that would cause a x % reduction in reproductive output, i.e. ELx (e.g. EL50, EL20, or EL10).
The survival of the parent animals and time to production of first brood must also be reported. Other substance-related effects on parameters such as growth (e.g. length), and possibly intrinsic rate of increase, may also be examined.
 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.
 OECD Guidelines for the Testing of Chemicals. 211. Daphnia magna Reproduction Test. Paris: OECD, Adopted 2 October, 2012.
 China National Technical Committee for Standardization of Dangerous Chemicals Management. GB/T 21828 - 2008, Chemicals Daphnia magna Reproduction Test. Beijing:China Standards Press, 2008.
 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: Daphnia magna
Exposure duration: 21d
Test concentration: According to the results of other tests, a nominal loading rate of 100mg/L water-accommodated fraction (WAF) solution test group and a blank control containing no test substance was selected.
Replicate: 10 replicates
Number of animals: 10 / test group
Test medium: Elendt M4 medium
Type of test: Semi-static test (renewal at every 24h)
Preparation of test solution: 0.500g test substance was obtained in a flask with a small amount of test medium then transferred into 5L-jar and diluted with test medium to 5000mL. And the test solution 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 from the middle and bottom layer was removed by siphoning into a beaker (the first approximated 100-200mL discarded) to give the nominal loading rate of 100mg/L WAF (Water-accommodated fraction) solution.
Preparation of test system: Elendt M4 medium was divided into 10 test vessels, respectively, and the volume of test medium is 80mL each test vessel as blank control group. The nominal loading rate of 100mg/L WAF test solution was divided into 10 test vessels, respectively, and the volume of test medium is 80 mL each test vessel as test substance group.
Volume of test solution: 80mL / test vessel
Temperature of test solutions: 19.4°C - 20.2°C
Photoperiod: 16h illumination / 8h darkness
Feeding: A concentrated algal suspension is fed to the Daphnia daily, the supplied diet was 0.1mg C/Daphnia/day - 0.2mg C/Daphnia/day.
Aeration: No aeration
Analysis of the actual concentrations of test substance in test solution: The actual concentrations of the nominal loading rate of 100mg/L WAF test group were measured at 0d, 5d, 9d, 13d, 16d, 20d (new solution) and 1d, 6d, 10d, 14d, 17d, 21d (old solution) by GC. The actual concentrations of blank control were measured at 0d and 1d (before the renewal) after the start of the test.
The measured concentrations of the test substance in test solutions: 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 100mg/L loading rate WAF test group were all lower than the limit of detection (LOD = 0.863 mg/L) at 0d, 5d, 9d, 13d, 16d, 20d in the new solution and at 1d, 6d, 10d, 14d, 17d, 21d in the old solution.
21d Lowest Observed Effect Loading rate (LOEL)
LOEL for effects on reproduction Greater than nominal loading rate of 100mg/L WAF
LOEL for effects on body length Greater than nominal loading rate of 100mg/L WAF
LOEL for mortality of parent animals Greater than nominal loading rate of 100mg/L WAF
21d No Observed Effect Loading rate (NOEL)
NOEL for effects on reproduction Nominal loading rate of 100mg/L WAF
NOEL for effects on body length Nominal loading rate of 100mg/L WAF
NOEL for mortality of parent animals Nominal loading rate of 100mg/L 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 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.
No classification is applicable.
Assessment of chronic toxicity to invertebrates
A Klimish 1 GLP study conducted in accordance with current OECD guidelines is available to assess the effects of acute toxicity to Daphnia Magna. The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 21-Day Lowest Observed Effect Loading rate value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF. 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.
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