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Long-term toxicity to fish

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Reference
Endpoint:
fish, juvenile growth test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 5 August 2016 and 5 September 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 215 (Fish, Juvenile Growth Test)
Deviations:
no
Principles of method if other than guideline:
Observations
The fish was examined daily during the test period and any external abnormalities (such as hemorrhage, discoloration) and abnormal behavior (such as inactivity, strange swimming pattern, other abnormal behaviour, etc.) were noted.
Fish were considered dead if there was no visible movement (e.g. gill movements) and if touching of the caudal peduncle produces no reaction. The dead fish were removed as soon as possible, if any.

Measurements
At the start and the end of the test, all surviving fish were weighed as wet weights (blotted dry) by individual.
Each freshly prepared treatment (day 0) and each old test medium (day 3, 9, 18, 28) were analyzed.
Measurements of pH, dissolved oxygen and temperature were carried out at the beginning and thereafter (0, 3, 9, 18, 28 d).
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) Reaction product of terephthalic acid, 2-ethylhexanol and 1-butanol (IUPAC name)
CAS: 1571954-81-8
(A:1962-75-0, B:1429441-82-6, C:6422-86-2)
Molecular formula: A : C16H22O4; B : C20H30O4; C : C24H38O4
Molecular weight: A: 278, B: 334, C: 390
Lot No.: GLFG160607
Purity: 99.8%
Exp. Date: 2017.06.01
Water solubility: 2.19 mg/L (20 oC)
Appearance: Colorless liquid
Storage conditions: Room temperature (25 oC),no contact with air and light
Analytical monitoring:
yes
Details on sampling:
10.0 mL collected water samples were taken from each freshly prepared (0 d) concentration and old medium (3, 9, 18, 28 d). After diluted with 10 mL acetonitrile, samples were filtered through 0.22 μm millipore membrane (discarding the initial 2.00 mL filtrates), then the filtrates were analysed by UPLC-PDA.
Vehicle:
no
Details on test solutions:
Test Concentrations
According to the information provided by the sponsor, the 96h-LL50 of the test substance to rainbow trout was over 100 mg/L loading rate WAF, so five test concentrations of 10 mg/L, 20 mg/L, 40 mg/L, 80 mg/L and 100 mg/L loading rate WAF were selected in the Juvenile Growth Test. Additionally, the blank control was tested in parallel (dilution medium without test substance).

Preparation of the Test Solutions
In the test, WAFs (water accommodated fractions) solutions were prepared by adding appropriate amount of the test substance to the 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 on a magnetic stirplate and a telfon stirbar at room temperature. The vortex height was set at least 10% of the static liquid height. At the end of the stirring, the mixture was allowed to settle for 2 hour 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 remaining undissolved test substance.
The details of the test solutions preparation were as follows:

Concentrations Amount of test substance added (g) Dilution Medium Volume (L)
Blank Control 0 3
10 mg/L WAFs 0.030 3
20 mg/L WAFs 0.060 3
40 mg/L WAFs 0.120 3
80 mg/L WAFs 0.240 3
100 mg/L WAFs 0.300 3
Test organisms (species):
other: Rare minnow (Gobiocypris rarus)
Details on test organisms:
The test species of Rare minnow (Gobiocypris rarus, Batch No.: JF20160615G) were bred by our own lab. A population of a single stock was used in the test. Details concerning the source of the fish were recorded in the raw data.
Feeding fry were held for 14 days in holding tanks supplied with a continuous flow of aerated water before being used for testing. During the pre-culture, the following conditions were maintained:
-Light: 16 hours photoperiod daily;
-Temperature: 23.1°C to 23.5°C for Rare minnow;
-Oxygen concentration: 90% ~ 96% of the air saturation;
-Feeding: The fish were fed daily with live food (Artemia) daily and the quantity of food being kept constant at 4% dry weight related to the initial fish weight. Food was withheld from the stock population for 24 h prior to the start of the test.
During the holding period the tanks were inspected daily and any debris or unhealthy or dead fish were removed. Fish received no treatment for disease in the two weeks preceding the test and during the test.
After the 48 h settling-in period, no mortality was observed in the following 7 days. So this batch of fish was accepted (Batch No.: JF20160615G).
Using a general anaesthetic (an aqueous solution of 100 mg/L tricaine methane sulphonate (MS 222) neutralized by the addition of two parts of sodium bicarbonate per part of MS 222), a subsample of 10 fishes before the test were weighed individually as wet weights (blotted dry) with the precision of 0.1 mg. The wet-weight of the acclimated batch of test fish at the start of test is showed in Table 8. Handling of juvenile fish was done with the utmost care to avoid stressing and injuring test animals.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
28 d
Post exposure observation period:
None
Hardness:
Not measured
Test temperature:
23.1 to 23.4 °C
pH:
7.69 to 7.92
Dissolved oxygen:
77 to 95%
Salinity:
Not measured
Conductivity:
Not measured
Nominal and measured concentrations:
According to the information provided by the sponsor, the 96h-LL50 of the test substance to rainbow trout was over 100 mg/L loading rate WAF, so five test concentrations of 10 mg/L, 20 mg/L, 40 mg/L, 80 mg/L and 100 mg/L loading rate WAF were selected in the Juvenile Growth Test. Additionally, the blank control was tested in parallel (dilution medium without test substance).

The mean of the measured concentrations were 2.93, 4.22, 4.65, 9.35 and 10.5 mg/L, respectively. The results indicated that concentration of test substance was stable (within 80% of the initial concentration) in the water during the test period. Thus semi-static conditions of 72 h-renewal used in the test was reasonable.
Details on test conditions:
In the test, WAFs (water accommodated fractions) solutions were prepared by adding appropriate amount of the test substance to the 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 on a magnetic stirplate and a telfon stirbar at room temperature. The vortex height was set at least 10% of the static liquid height. At the end of the stirring, the mixture was allowed to settle for 2 hours 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 remaining undissolved test substance.
The details of the test solutions preparation were as follows:

Concentrations Amount of test substance added (g) Dilution Medium Volume (L)
Blank Control 0 3
10 mg/L WAFs 0.030 3
20 mg/L WAFs 0.060 3
40 mg/L WAFs 0.120 3
80 mg/L WAFs 0.240 3
100 mg/L WAFs 0.300 3

Conditions of Exposure
Depend on the stability of the test solution, 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 condition.
During the test, the following conditions were maintained:

Duration: 28 days.
Tanks: Made of chemically inert material, with a sealable inert lid, and with a capacity of approximately 5 L.
Volume of the test solution: 3 L.
Loading: 0.2 to 0.4 g of fish/L.
Stocking densities: 10 for each treatment and control.
Replicates: none.
Light: 16-hour light and 8-hour dark cycle daily.
Temperature: 23.1°C ~ 23.4°C for Rare minnow.
Oxygen concentration: Not less than 60 per cent of the maximum air saturation value throughout the test; aeration forbidden.
Feeding: daily; Quantity of food being kept constant at 4% dry weight related to the initial fish weight.
Cleaning: remove residual fish food in the solution daily with siphon. The tank will be replaced by a clean one each time the solution renewal.
Reference substance (positive control):
no
Key result
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: WAF
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: WAF
Details on results:
Mortality and Effects

The results showed that under valid semi-static test conditions (72 h-renewal), no fish died in control and treatments groups. Fish in all treatments and control groups were observed alive and appeared normal.
At the beginning of the test, the average weight of the fish in control is 0.0579 g, and 0.155 g at the end of the test. The mean weight of fish in the controls increased more than 50%. Based on analysis of variance, the pseudo specific growth rates for each concentration were then compared with the control values using the Bartlett’s test (ANOVA, STATA 10.0). The results showed that there was no significant difference for pseudo specific growth rates in 10.0, 20.0, 40.0, 80.0 and 100 mg/L WAFs treatments groups compared with the blank control group (p > 0.05). Therefore the LOEC for the growth rate of the test substance to rare minnow is more than 100 mg/L WAFs (measured concentration 10.5 mg/L with no filtration, 0.47 mg/L after filtration), the NOEC is equal to 100 mg/L WAFs (measured concentration 10.5 mg/L with no filtration, 0.47 mg/L after filtration).
Results with reference substance (positive control):
N/A
Validity criteria fulfilled:
yes
Conclusions:
The results showed that under valid semi-static test conditions (72 h-renewal), in the 28 d test period, the LOEC (lowest observed effect concentration) and the NOEC (No observed effect concentration) for the growth rate of the test substance to rare minnow were concluded as follows:
28 d-LOEC > 100 mg/L WAFs (measured concentration 10.5 mg/L with no filtration, 0.47 mg/L after filtration);
28 d-NOEC = 100 mg/L WAFs (measured concentration 10.5 mg/L with no filtration, 0.47 mg/L after filtration).
Executive summary:

Under semi-static conditions of72 h-renewal, the effect on growth rates and other observed effects in juvenile fish (Gobiocypris rarus) exposed to the test substance for 28 days was conducted according to the following guidelines: “The guidelines for the testing of chemicals” (HJ/T 153-2004),“The Guidelines for the Testing of Chemicals, Effects on Biotoc Systems”(the2ndedition)(2013);Procedure 215of the “Guidelines for Testing of Chemicals”of the OECD: “Fish, Juvenile Growth Test” (2000) etc.

Nominal concentrations of 10, 20, 40, 80 and 100mg/LWAFs test solutions were used in test. During the whole test period, the pH values of the control and test media were between 7.69 and 7.92, and the Dissolved Oxygen (DO) values varied from 77% ~ 95% of the air saturation at the test temperature, and the temperature of the test media was maintained at 23.1 °C ~ 23.4 °C. All fishes in the control group were normal. The mean weight of fish in the control group increased more than 50%. So the study met the acceptability criteria prescribed by the protocol (dissolved oxygen concentration: no less than 60% of the air saturation value; temperature: (23±2)ºC and not differ by more than ±1ºC between test chambers; the increasing rate of the mean wet-weight of fish: no less than 50% of the initial weight). Therefore the test is valid.

In order to confirm the stability of the test substance in the test medium, concentrations of the test samples during the test period were analysed by UPLC-PDA. As the results of the standard solution analysis, a linear regression equation was obtained with the peak area values vs. the concentration of GL500 (0, 0.20, 0.50, 1.00, 2.00, 5.00 and 10.0 mg/L) A=100305c-235.81, with good linearity ofr2= 1. The result showed that linearity for the GL500 with the concentration range of 0.20 mg/L to 10.0 mg/L was good.The mean of the measured concentrations were 2.93, 4.22, 4.65, 9.35 and 10.5 mg/L, respectively. The analytical results showed that the concentration of the test substance of the test solution was stable throughout a period of 72 h (deviation within 20%). Thus a semi-static test of 72 h-renewal was reasonable.

The results showed that under valid semi-static test conditions (72 h-renewal), all fish in the control groups and treated groups (10, 20, 40, 80 and 100mg/LWAFs) were alive and appeared normal. In the 28d test period, the LOEC of the test substance for the juvenile fish growth rate is greater than 10 mg/L WAFs (measured concentration 10.5 mg/Lwith no filtration, 0.47 mg/L after filtration), and NOEC is equal to 10.0 mg/L WAFs (measured concentration 10.5 mg/L with no filtration, 0.47 mg/L after filtration), thus:

28 d-LOEC >10.0 mg/L WAFs (measured concentration 10.5 mg/Lwith no filtration, 0.47 mg/L after filtration);

28 d-NOEC=10.0 mg/L WAFs (measured concentration 10.5 mg/Lwith no filtration, 0.47 mg/L after filtration).

Description of key information

The results showed that under valid semi-static test conditions (72 h-renewal), in the 28 d test period, the LOEC (lowest observed effect concentration) and the NOEC (No observed effect concentration) for the growth rate of the test substance to rare minnow were concluded as follows:

28 d-LOEC > 100 mg/L WAFs (measured concentration 10.5 mg/L with no filtration, 0.47 mg/L after filtration);

28 d-NOEC = 100 mg/L WAFs (measured concentration 10.5 mg/L with no filtration, 0.47 mg/L after filtration).

Key value for chemical safety assessment

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

Additional information