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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 23 August 2016 and 22 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:

Chemical name: 1,4-Benzenedicarboxylic acid, mixed Bu and 2-ethylhexyl diester (CAS name)
CAS: 1571954-81-8(A: 1962-75-0, B: 1429441-82-6, C: 6422-86-2)
Chemical formula: A:C16H22O4, B:C20H30O4, C:C24H38O4
Molecular weight: A:278, B:334, C:390
Purity/Assay: 99.8%
Water solubility: 2.19 mg/L (20°C)
Lot No: GLFG160607
Expiry date: 2017-06-01
Appearance: Colorless liquid
Storage conditions: Room temperature (25°C),no contact with air and light
Analytical monitoring:
yes
Details on sampling:
Two parallels of 10.0 mL WAFs water samples were taken from 10mg/L test solution at the beginning (0 d), immediately prior to the first renewal of the test solution (3 d) and weekly thereafter (9, 21 d). One parallel 10.0 mL samples at 10.0 mg/L WAFs concentration was filtrated by 0.22 µm membrane (discarding the initial 2.00 mL filtrates), The other was diluted 1 times with 10.0 mL acetonitrile and filtrated by 0.22 µm membrane (discarding the initial 2.00 mL filtrates) , then the filtrates analysed by the above UPLC-PDA method.
Vehicle:
no
Details on test solutions:
Elendt M7 was used as the control and test media.

In the range-finding test, animals were exposed to concentrations of 1.00 mg/L WAFs and 10.0 mg/L WAFs. A blank control was tested in parallel (dilution medium without test substance).
Based on the results of the range-finding test, nominal concentration of 10.0 mg/L WAFs were conducted in limit test . Additionally, one control group were tested in parallel (dilution medium without test substance).

Preparation of the Test Solutions
WAFs (water accommodated fractions) solutions with a nominal concentration of 1.00 mg/L were produced by adding 0.0012 g of the test substance to the 1000 mL Elendt M7 test medium in glass aspirator bottles with appropriate size. The mixing aspirator bottle was stoppered and the aqueous test substance mixture was stirred for 71 hrs at room temperature (about 23°C - 25°C). The vortex height was set at least 10% of the static liquid depth. At the end of the 71 h, stirring was stopped and the mixture was allowed to settle for 2 h at room temperature. Then the aqueous portion removed through the outlet of the aspirator stirring bottle was the WAFs solutions of the test substance without further filtration to remove the floating undissolved test substance.

WAFs (water accommodated fractions) solutions with a nominal concentration of 10.0 mg/L were produced by adding 0.0097 g of the test substance to the 1000mL Elendt M7 test medium in glass aspirator bottles with appropriate size. The mixing aspirator bottle was stoppered and the aqueous test substance mixture was stirred for 71 hrs at room temperature (about 23°C - 25°C). The vortex height was set at least 10% of the static liquid depth. At the end of the 71 h, stirring was stopped and the mixture was allowed to settle for 2 h at room temperature. Then the aqueous portion removed through the outlet of the aspirator stirring bottle was the WAFs solutions of the test substance without further filtration to remove the floating undissolved test substance.

50.0 mL WAFs test solutions at different concentrations were added to glass beakers. Details were described as follows:

Test Type Nominal Concentration Amount of test substance Added (g) Total Volume (L)
(mg/L)
Range-finding Test Blank control — 1
1.00 (WAFs) 0.0012 1
10.0 (WAFs) 0.0097 1
Limit Test Blank control — 1
10.0 (WAFs) 0.0102 1
Test organisms (species):
Daphnia magna
Details on test organisms:
Daphnia magna was introduced from Nanjing University in 2010 and culture medium was Elendt M7. Daphnia magna, not being the first brood progeny, was the test species and were 6 to 24 hours old at the start of the test. All organisms used for a particular test originated from cultures established from the same healthy stock of Daphnia. The stock animals were maintained in culture conditions (light, temperature, and medium) similar to those to be used in the test, Lot No for range-finding test and limit test were D20160908D and D20160929D.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Post exposure observation period:
None
Hardness:
221 to 232 mg (CaCO3)/L
Test temperature:
20.3 to 21.1 ºC
pH:
7.46 and 7.80
Dissolved oxygen:
90% to 101%
Nominal and measured concentrations:
In the range-finding test, animals were exposed to concentrations of 1.00 mg/L WAFs and 10.0 mg/L WAFs. A blank control was tested in parallel (dilution medium without test substance).
Based on the results of the range-finding test, nominal concentration of 10.0 mg/L WAFs were conducted in limit test . Additionally, one control group were tested in parallel (dilution medium without test substance).

The analytical results from the acetonitrile diluted group show that the analysed concentrations were consistent in the test medium throughout the test period (deviation within 20% of the initial concentration). Thus a semi-static method of 72h renewal test procedure was reasonable.
Details on test conditions:
The concentration of the test substance was demonstrated to be maintained within ±20 percent of the measured initial concentration throughout the test under a 72 h renewal conditions. When the medium was renewed, a second series of test vessels were prepared and the parent animals were then be transferred to them by a glass pipette of suitable diameter. All the beakers lids were put on.The volume of medium transferred with the Daphnia was minimized.
Ten animals were individually held at each test concentration and in the control. Treatments were allocated to the test vessels and all subsequent handling of the test vessels was done in a random fashion. The test conditions were maintained as follows:
-Light: 16-hour light and 8-hour dark cycle daily (light intensity: 1000 to 1500 lux);
-Temperature: 20.3~21.1ºC;
-Oxygen concentration: 90% to 101%; no aerating;
-pH: 7.46~7.80;
-Feeding: Chlorella vulgaris Beij. was given once every day, 0.1 - 0.2 mg C/Daphnia each time.
-Aeration: None.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
mortality
Remarks:
Survival
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
mortality
Remarks:
Survival
Details on results:
During the test, all animals in the blank control were alive and the number of live offspring produced per parent animal surviving at the end of the test was 61~83. The coefficient of variation around the mean number of living offspring produced per parent animal in the control(s) was 8.82% (<25%). At the end of the test period, no adults died in blank control and 10.0 mg/L WAFs treatments. Thus the LOEC for survival of adults was more than 10.0 mg/L WAFs (the measured concentration 3.33 mg/L) and the NOEC was 10.0 mg/L WAFs (the measured concentration 3.33 mg/L).

According to the Independent Samples T-test analysis results of the test data (Independent Samples T-test, SPSS 17.0), the significance level of p was more than 0.05. Thus the reproduction rates in the treatments of 10.0 mg/L WAFs treatments showed no statistically significant difference from that of the blank control (p>0.05). Thus the LOEC for the Daphnia magna reproduction was more than 10.0 mg/L WAFs (the measured concentration 3.33 mg/L) and the NOEC was 10.0 mg/L WAFs (the measured concentration 3.33 mg/L).
Validity criteria fulfilled:
yes
Conclusions:
Under valid semi-static test conditions (72h-renewal), for Daphnia magna adult survival and reproduction the LOEC was more than 10.0 mg/L WAFs (the measured concentration 3.33 mg/L) and the NOEC was 10.0 mg/L WAFs (the measured concentration 3.33 mg/L). For survival of adults the LOEC was more than 10.0 mg/L WAFs (the measured concentration 3.33 mg/L), the NOEC was more than 10.0 mg/L WAFs (the measured concentration 3.33 mg/L ). i.e.:
LOEC (Reprodution) > 10.0 mg/L WAFs (the measured concentration 3.33 mg/L );
NOEC (Reprodution) = 10.0 mg/L WAFs (the measured concentration 3.33 mg/L) ;
LOEC (Survival) > 10.0 mg/L WAFs (the measured concentration 3.33 mg/L);
NOEC (Survival) =10.0 mg/L WAFs (the measured concentration 3.33 mg/L).
Executive summary:

Under semi-static conditions of 72h renewal,the effect on the reproductive output of Daphnia magna exposed to the test substance (GL500) for 21 days was conducted according to “The guidelines for the testing of chemicals-effects on biotic systems” (HJ/T 153); , “The Guidelines for the Testing of Chemicals" (the 2nd edition,2013); Procedure 211 of the Daphnia magna, Reproduction Test”, OECD Guidelines for Testing of Chemicals, 211, adopted on 2 October 2012.

During the test period, the pH values of the control and test media were between 7.46 and 7.80, the Dissolved Oxygen (DO) values varied from 90% to 101% of the air saturation at the test temperature, the hardness values varied from 221 to 232 mg(CaCO3)/L, and the temperature of the control and test media was maintained in the range of 20.3oC to 21.1oC. All animals in the control were alive and the number of live offspring produced per parent animal surviving at the end of the test was61~83. The coefficient of variation around the number of living offspring produced per parent animal in the control(s) was8.82%. Therefore, the study met the acceptability criteria and was considered valid.

The concentration ofthetest substance (GL500) in test medium was determined by UPLC-PDA to represent the stability of the test substance in test period. A linear regression equation was obtained with the mean peak squares (A) vs. the concentration of the GL500 (c,mg/L): A=100182c-214.53with good linearity of r2=1.The results show that linearity for the concentration range of 0 mg/Lto 10.0 mg/Lwas good. The minimum detection amount of UPLC-PDA was0.06 mg/L and the minimum detection concentration of this method was 0.40 mg/L with dilution 2 times by acetonitrile. The analytical results showed that the concentration of the test substance was consistent in the test medium throughout the test period (deviation within 20% of the initial concentration). Thus a semi-static method of 72h renewal test procedure was reasonable.

The results showed that under valid semi-static test conditions (72h-renewal), for Daphnia magna reproduction and adult survival, the Lowest Observed Effect Concentration (LOEC) was more than 10.0 mg/L WAFs (the measured concentration3.33 mg/L) and the No Observed Effect Concentration (NOEC)was 10.0 mg/L WAFs (the measured concentration3.33 mg/L). For survival of adults the LOEC was more than 10.0 mg/LWAFs (the measured concentration 3.33 mg/L), the NOEC was 10.0 mg/LWAFs (the measured concentration3.33 mg/L).

Description of key information

Under valid semi-static test conditions (72h-renewal), for Daphnia magna adult survival and reproduction the LOEC was more than 10.0 mg/L WAFs (the measured concentration 3.33 mg/L) and the NOEC was 10.0 mg/L WAFs (the measured concentration 3.33 mg/L). For survival of adults the LOEC was more than 10.0 mg/L WAFs (the measured concentration 3.33 mg/L), the NOEC was more than 10.0 mg/L WAFs (the measured concentration 3.33 mg/L ). i.e.:

LOEC (Reprodution) > 10.0 mg/L WAFs (the measured concentration 3.33 mg/L );

NOEC (Reprodution) = 10.0 mg/L WAFs (the measured concentration 3.33 mg/L) ;

LOEC (Survival) > 10.0 mg/L WAFs (the measured concentration 3.33 mg/L);

NOEC (Survival) =10.0 mg/L WAFs (the measured concentration 3.33 mg/L).

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
10 mg/L

Additional information