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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:

10.2020 - 11.2020

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

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.

Version / remarks:

2004

Qualifier:

according to guideline

Guideline:

other: ASTM international. Standard Practice for Aquatic Toxicity Testing of Lubricants: Sample Preparation and Results Interpretation. D6081 − 19 (Reapproved 2019).

Version / remarks:

2019

Qualifier:

according to guideline

Guideline:

other: ECETOC. Aquatic Toxicity Testing of Sparingly Soluble, Volatile and Unstable Substances [S].ECETOC Monograph No. 26 1996.

Version / remarks:

1996

Qualifier:

according to guideline

Guideline:

other: OECD. Series on testing and assessment No. 23 (Second Edition) Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals. Paris: OECD, Adopted Feburuary 8, 2019.

Version / remarks:

2019

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2013

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

Elendt M4 medium was prepared with deionized water

Test organisms (species):

Daphnia magna

Details on test organisms:

(1) Species
Daphnia magna
(2) 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 ".
(3) 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 DMU20200930-1.
(4) 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 lx -1500 lx). During the acclimation, Daphnia magna were fed with a concentrated algal suspension.
(5) Size
The daphnids were less than 24 hours old and were not first brood progeny. The female Daphnia magna were used in the test.
(6) Number
20
(7) Allocation to the test groups
The daphnids were allocated at random to each test group.
4.2 Food
(1) Species
Pseudokirchneriella subcapitata
(2) Supplier
Committee on Type Culture Collection of Chinese Academy of Sciences.

(3) Condition of culture
Temperature (20 °C ± 2 °C), 24 h continuously light, illumination intensity (4440 lx-
8880 lx).

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21

Nominal and measured concentrations:

15 mg/L

Details on test conditions:

Test conditions
Test substance LICOCARE RBW 300 FL TP
Test organism Daphnia magna
Exposure duration 21 days (d)
Test concentration According to the results from preliminary test (see Annex B), the limit test was performed with a nominal loading rate of 15.0 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 0.030 g test substance was weighed and slowly added into a 2 L beaker which contained 2000 mL Elendt M4 medium, and the nominal concentration of 15.0 mg/L test suspension was obtained and stirred continuously with a magnetic stirrer for about 48 h. The vortex of stirring intensity was maintained at about 10% of liquid height. After stirring, the solution was allowed to stand for about 24 hour, then 1000 mL of middle layer solutions was siphoned into another beaker (the first 100 mL was discarded), the nominal loading rate of 15.0 mg/L WAF solution was obtained and used as test solution.
Preparation of test system 15.0 mg/L WAF solution was divided into 10 test vessels, and the volume of test solution 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 19.9 °C – 21.5 °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 total organic carbon (TOC) in test solutions
The actual concentrations of TOC in blank control and 15.0 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 Vario Toc analyzer.

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

8 Results
8.1 Observation of test solutions
After standing, the WAF solution in the middle layer was collected into another test vessel as test solution. The WAF solution was checked through Tyndall effect by a laser beam, a small amount of fine particles were observed in the light path, The Tyndall effect was slightly positive. During the test period, the blank control solutions were all observed to be clear and colorless.
8.2 Observations of parent daphnids
The result is shown in Table 1 and Table 2.
During test period, there was no abnormal color and responses of parent daphnids in blank control and the 15.0 mg/L WAF test group.
The time to the presence of the first eggs in the brood pouch in blank control and 15.0 mg/L WAF test group were 7.0 ± 0.0 d and 7.0 ± 1.0 d respectively, and the time to production of first brood in blank control and 15.0 mg/L WAF test group were 9.9 ± 0.4 d d and 9.9 ± 0.9 d respectively.
8.3 Mortality rate of parent daphnids
The result is shown in Table 3.
At the end of the test, there was no parent daphnids dead in the blank control group, which met the validity criteria.
At the end of the test, the mortality of parent daphnids in the 15.0 mg/L WAF test group was 0%. So according to the mortality of parent daphnid, LOEL was greater than the nominal loading rate of 15.0 mg/L (WAF), NOEL was the nominal loading rate of 15.0 mg/L (WAF).
8.4 Body length of the parent daphnids
The mean body length and the standard variance of the parent daphnids in each group are shown in Table 4.
Results of T-test shown that, there was no significant different (p>0.05) on body length of parent animals between the blank control and 15.0 mg/L WAF test group. So according to the body length of the parent daphnids, LOEL was greater than the nominal loading rate of 15.0 mg/L (WAF), NOEL was the nominal loading rate of 15.0 mg/L (WAF).
8.5 Living offspring by each parent daphnid
The results are shown in Tables 5, 6 and figure 1.
At the end of the test, the mean ± Standard Deviation (SD) of reproductive output of the blank control group was 102 ± 6 offspring per surviving parent daphnid. The coefficient of variation in the control group was 6%.
No dead offspring and aborted broods were observed in blank control and 15.0 mg/L WAF test group during the test.
The living offspring produced per surviving parent daphnid in each test group were estimated by T-test. Results showed that there was no significant different (p>0.05) on number of living offspring between the blank control and 15.0 mg/L WAF test group. So according to the reproductive output, LOEL was greater than the nominal loading rate of 15.0 mg/L (WAF), NOEL was the nominal loading rate of 15.0 mg/L (WAF).
8.6 Measurements of illumination and water temperature, pH, DO and hardness
The results are shown in Table 7 and Table 8 and figure 2.
The average illumination intensity at the start and the end of the test were 1291 lx and 1288 lx, respectively. During the test, the result of continuous monitoring of the water temperature was shown that the water temperature was 19.9 °C – 21.5 °C.
The pH, DO and hardness of blank control and 15.0 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 7.96 - 8.84, DO was 8.2 mg/L - 9.2 mg/L, hardness was 240 mg/L - 265 mg/L (as CaCO3).

8.7 Measurement of TOC concentrations in test solutions
8.7.1 Sample pretreatment and preservation
The test solution of blank control and 15.0 mg/L WAF were filtered with 0.45 μm aqueous phase polyether sulfone (PES) filter membrane (the first 5 mL - 10 mL filtrates were discarded) before analysis. The samples were analyzed immediately, if not, the samples were stored at -20°C, and the measurement was carried out within 7 d.
8.7.2 The results of the measurement of TOC concentrations
The limit of detection (LOD) and the limit of quantification (LOQ) of TOC were 0.2 mg/L and 0.8 mg/L, respectively. The linearity range of this test was 0.80 mg/L to 50.0 mg/L, the specific results are shown in Annex C.
The measured concentrations of TOC in blank control on 0 d, 7 d, 14 d (new solution) and 1 d, 8 d, 15 d (old solution) ranged from <0.2 mg/L to <0.8 mg/L.
The measured concentrations of TOC in 15.0 mg/L WAF test group on 0 d, 7 d, 14 d (new solution) and 1 d, 8 d, 15 d (old solution) ranged from <0.2 mg/L to 0.942 mg/L, which were close to the blank control’s, indicating that test substance was poorly soluble in test water.

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 15.0 mg/L (WAF)
LOEL for effects on body length > nominal loading rate of 15.0 mg/L (WAF)
LOEL for mortality of parent daphnids > nominal loading rate of 15.0 mg/L (WAF)

21d No Observed Effect Loading rate (NOEL)
NOEL for effects on reproduction = nominal loading rate of 15.0 mg/L (WAF)
NOEL for effects on body length = nominal loading rate of 15.0 mg/L (WAF)
NOEL for mortality of parent daphnids = nominal loading rate of 15.0 mg/L (WAF)

Executive summary:

(1) Basis of determined concentration

LICOCARE RBW 300 FL TP is a kind of UVCB substance with low water solubility. And it’s not soluble in regular solvent (Methanol, acetonitrile, tetrahydrofuran, isopropanol, etc), so HPLC (High Performance Liquid Chromatograph) and LC-MS/MS (Liquid Chromatograph-Mass Spectrometer) is not suitable for analysis. According to the GC (Gas Chromatography) method in Certificate of Analysis that sponsor provided, the test substance should be derivated first, but derivatization must be proceeded under non-aqua system, which is not suitable for this test system and the analysis of water solution, meanwhile, the predicted limit of quantification (LOQ) of the analysis method would be high. Moreover, the carbon chain of some component of the test substance is much more than 40, so it’s not guaranteed that all constituents of test substance would be peaked in GC condition because of the high boiling point, and it would cause residual and pollute the apparatus. Consequently, in this test, only the concentrations of TOC in test solutions were measured to indicate the stability of exposed concentration of the test system

(2) Basis of the preparation of test solution

Since the test substance is a kind of UVCB substance with low water solubility, according to the references “Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals” (OECD number 23 Second Edition, February 8, 2019). “Standard Practice for Aquatic Toxicity Testing of Lubricants: Sample Preparation and Results Interpretation” (ASTM international, D6081 − 19), the test solutions will be prepared with the method of Water Accommodated Fraction (WAF). Given that the toxicity cannot be attributed to a single component but to LICOCARE RBW 300 FL TP as a whole, the test results are reported based on the nominal loading rates for the whole test substance.

(3) Measured concentrations of TOC in test solution

The measured concentrations of TOC in blank control on 0 d, 7 d, 14 d (new solution) and 1 d, 8 d, 15 d (old solution) ranged from <0.2 mg/L (LOD) to <0.8 mg/L (LOQ).

The measured concentrations of TOC in 15.0 mg/L WAF test group on 0 d, 7 d, 14 d (new solution) and 1 d, 8 d, 15 d (old solution) ranged from <0.2 mg/L to 0.942 mg/L, which were close to the blank control’s, indicating that test substance was poorly soluble in test water.

(4) 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:

 

21d Lowest Observed Effect Loading rate (LOEL)

LOEL for effects on reproduction         > nominal loading rate of 15.0 mg/L (WAF)

LOEL for effects on body length          > nominal loading rate of 15.0 mg/L (WAF)

LOEL for mortality of parent daphnids  > nominal loading rate of 15.0 mg/L (WAF)

 

21d No Observed Effect Loading rate (NOEL)

NOEL for effects on reproduction        = nominal loading rate of 15.0 mg/L (WAF)

NOEL for effects on body length          = nominal loading rate of 15.0 mg/L (WAF)

NOEL for mortality of parent daphnids = nominal loading rate of 15.0 mg/L (WAF)

Description of key information

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 15.0 mg/L (WAF)
LOEL for effects on body length > nominal loading rate of 15.0 mg/L (WAF)
LOEL for mortality of parent daphnids > nominal loading rate of 15.0 mg/L (WAF)

21d No Observed Effect Loading rate (NOEL)
NOEL for effects on reproduction = nominal loading rate of 15.0 mg/L (WAF)
NOEL for effects on body length = nominal loading rate of 15.0 mg/L (WAF)
NOEL for mortality of parent daphnids = nominal loading rate of 15.0 mg/L (WAF)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Remarks:

Limit concentration

Effect concentration:

15 mg/L