1,2,4-Benzenetricarboxylic acid, mixed lauryl and octyl triesters

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:

2015-04-01 - 2015-11-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

GLP compliance:

yes

Specific details on test material used for the study:

N/A

Analytical monitoring:

yes

Details on sampling:

The samples were analyzed directly without storage.

Vehicle:

no

Details on test solutions:

Elendt M4 medium was prepared by analytical reagent and deionized water and stored at 2 to 8 °C. The concentration of TOC in the medium (i.e. before addition of the algae) was measured regularly every year and it was 1.74 mg/L. The test medium was aerated for more than 30 minutes prior to the start of the test and placed in testing temperature at least 12h before the test. For the medium at the beginning and during the test, the dissolved oxygen concentration was 8.81-9.44mg/L; the pH was 7.79-8.35; the hardness was 165-175 mg/L(as CaCO3).

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain: Daphnia magna Straus
- Source: supplied by MicroBioTests lnc.and not first brood progeny were used at the start of the test.
- Age at study initiation: not older than 24h
- Method of breeding: Adult Daphnia magna were bred in the laboratory of APM in the Elendt M4 medium. The culture conditions were similar to test conditions.
- Feeding during test: Cultures of adult Daphnia magna were fed concentrated freshwater suspensions of unicellular green algae Desmodesmus Subspicatus and Pseudokirchneriella subcapitata at a rate equivalent to 0.1-0.2mg of organic carbon per adult per day.


ACCLIMATION
- Type and amount of food: concentrated freshwater suspensions of unicellular green algae Desmodesmus Subspicatus and Pseudokirchnerie/la subcapitata five times per week at a rate equivalent to 0.20mg of organic carbon per adult per day.
- Feeding frequency: daily

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Remarks on exposure duration:

N/A

Post exposure observation period:

none

Hardness:

165-175 mg/L CaCO3

Test temperature:

20 - 20.8 °C

pH:

7.79-8.35.

Dissolved oxygen:

8.81-9.44 mg/L

Salinity:

according to guideline (M4 medium, Elendt)

Conductivity:

N/A

Nominal and measured concentrations:

Nominal: 100 mg/L (saturated solution of the test substance)
Measured: < LOD (0.214 mg/L)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): illumination incubabor with 75 ml glass containers
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): 3 times per week (on the days 0, 2, 5, 7, 9, 12,1 4, 16, 19, 21 d)
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Biomass loading rate: not mentioned

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Synthetic fresh water according to Elendt M4-Medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: Day 0 (fresh samples), 2, 5, 7, 12, 14 d


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16h light, 8h dark
- Light intensity: not exceeding 1125-1326 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
mortality and offspring on day 2, 5, 7, 9, 12, 14, 16, 19, 21


TEST CONCENTRATIONS
- Range finding study:
The test substance is insoluble. 2 L solution of the test substance, the nominal concentration of which was 100 mg/L, was prepared with Elendt M4 medium and stirred (via magnetic stirrer) in the dark for 72 h at 500 rpm and were filtrated with 47 mm glass microfiber filters and 0.45 µm cellulose nitrate membrane filters to make a saturated solution of the test substance in the test medium as the stock solution and the test solutions of the highest concentration group. The test solutions were prepared once every week and subpackage stored at room temperature for 7 days. The pH of the stock solution measured before the test was 7.77 and needed not to be adjusted. Three treatment groups and one test-medium control series were included in the range-finding test. The nominal concentrations of the treatment group were 1%, 10% and 100% saturation solution. 5 animals individually held were tested for each test concentration. Each test concentration series consisted of replicates filled with test solutions of the same concentration. One animal was maintained individually in each replicate. The duration of the range-finding test was 21 days. The test vessels were not aerated during the test. The range-finding test was performed in the illumination incubabor with 75 mL glass containers and the ratio of light to dark was 16 h to 8 h. Parent animals were maintained individually, one per test vessel, with about 75mL of medium in each vessel.
A semi-static test was conducted. The frequency of medium renewal was three times per week in the range-finding test. The medium was renewed on the days 0, 2, 5, 7, 9, 12, 14, 16, 19d of the test. Every adult was fed concentrated freshwater suspensions of unicellular green algae Desmodesmus Subspicatus and Pseudokirchneriella subcapitata five times per week at a rate equivalent to 0.20mg of organic carbon per adult per day. The results of the observations made during the test were recorded five times per week. The offspring produced by each parent animal were removed and counted from the appearance of the first brood. The numbers of living offspring, aborted eggs and dead offspring were counted and recorded. Mortality among the parent animals was recorded at the same times as offsprings were counted. During the test, when the medium was renewed in the semi-static test, a second series of test vessels had been prepared and the parent animals were transferred to them by a glass pipette of suitable diameter.

The mortality of the parent animals in the control group was 0%. The mortality of the parent animals in the treatment groups was varied from 0% to 20%. According to the result of the Steel's Many-One Rank Test, the 21d NOEC for cumulative adult deaths of the test substance to Daphnia magna was the concentration of the 100% saturated solution in the test medium and the LOEC was higher than the concentration of the 100% saturated solution in the test medium. The death of the parent animals in all treatment groups was not related to the effect of the test substance. According to the result of the Dunnett's Test, the 21d NOEC for reproduction of the test substance to Daphnia magna was the concentration of the 100% saturated solution in the test medium and the LOEC was higher than the concentration of the 100% saturated solution in the test medium. Based on the result of range-finding test, a limit test was carried out.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

N/A

Results with reference substance (positive control):

N/A

Reported statistics and error estimates:

Dunnett's Test.
Statistical analysis of the data was accomplished with Toxcalc v5.0.32.

Table #1: Number of live offspring and the coefficient of variation for control fecundity

Nominal concentration groups of test substance	Day	1	2	3	4	5	6	7	8	9	10
The control	8	5	0	4	4	1	0	3	1	3	7
	9	7	6	9	11	3	4	7	0	6	10
	12	17	14	16	19	10	16	17	7	13	20
	13	20	21	8	19	19	14	23	17	26	19
	14	0	0	0	0	0	1	0	1	0	0
	15	0	0	0	0	0	0	0	0	0	0
	16	17	19	15	17	16	13	12	15	15	14
	19	34	28	28	28	31	24	29	33	26	29
	20	0	0	0	0	0	0	1	0	0	0
	21	0	0	0	0	0	0	0	0	0	0
	Total	100	88	80	98	80	72	92	74	89	99
	Mean	87
	CV (%)	12 %
The 100 % saturated solution	9	3	0	4	4	0	0	0	0	0	0
	12	11	16	13	10	13	10	13	11	9	11
	13	15	25	18	0	21	18	18	16	12	18
	14	0	0	0	0	1	0	3	3	1	0
	15	0	0	0	0	0	0	0	0	0	0
	16	18	20	12	0	19	14	18	15	0	17
	19	30	32	31	0	31	26	30	26	4	26
	20	0	0	0	0	0	0	0	0	0	0
	21	0	27	38	0	30	26	32	32	0	31
	Total	77	120	116	14	115	94	114	103	26	103
	Mean	88
	CV (%)	43 %

Validity criteria fulfilled:

yes

Conclusions:

Expressed as the concentration of the 100% saturated solution in the test medium, the effect of 1,2,4-BENZENETRICARBOXYLIC ACID, MIXED DODECYL AND OCTYL TRIESTERS on Daphnia magna reproductive output is as following: The 21d No Observed Effect Concentration (NOEC) was not less than the concentration of the saturation solution in the test medium (the measured concentration of the 100% saturated solution was below the limit of detection (LOD) and the LOD was 0.214mg/L); The 21d Lowest Observed Effect Concentration (LOEC) was higher than the concentration of the saturation solution in the test medium (the measured concentration of the 100 % saturated solution was below the limit of detection (LOD) and the LOD was 0.214 mg/L); The 21d EC50 (50% reduction of the reproductive output) was higher than the concentration of the saturation solution in the test medium (the measured concentration of the 100% saturated solution was below the limit of detection (LOD) and the LOD was 0.214mg/L). The mortality of the parent animals in control group was 0%, not exceed 20%, and the mortality of the parent animals in the treatment group was 0% at the end of the test. No toxicity was observed under test conditions in the range of water solubility.

Executive summary:

According to "Ministry of environmental protection of the people's Republic of China, The Guidelines for the Testing of Chemicals - Effects an Biotic Systems, 2013, 211 Daphnia magna Reproduction Test" and "OECD, Guidelines for the Testing of Chemicals, 211 Daphnia magna Reproduction Test, 2012", the effects of the test substance 1,2,4-BENZENETRICARBOXYLIC ACID, MIXED DODECYL AND OCTYL TRIESTERS to Daphnia magna was determined in a 21 days reproduction test. The frequency of medium renewal was three times per week. Based an the results of the range-finding test, one treatment group and a control group were included in the limit test. A nominal concentration of 100 mg/L of the test substance was prepared and stirred (via magnetic stirrer) in the dark for 72h at 500 rpm followed by filtration using a 47 mm glass microfiber filters and 0.45 µm cellulose nitrate membrane filters to make a saturated solution of the test substance in the test medium as the test solution (referred to as "100 % saturated solution" in this report) of the treatment group. The test solutions were prepared once every week and stored at room temperature for 7 days.

 

The mortality of the parent animals in control group was 0%, not exceed 20%, and the mortality of the parent animals in the treatment group was 0 % at the end of the test. The mean number of live offspring produced per parent animal surviving at the end of the test in control group was 87, not less than 60, and the coefficient of variation of which was 12 %, not exceed 25 %.The test is valid.

The concentrations of the test substance in the test solutions were analyzed by using GC method. The measured concentration of the 100% saturated solution was below the limit of detection (LOD) in the preliminary test and the LOD was 0.214mg/L. Therefore the results were expressed as the concentration of the 100 % saturated solution.

 

Results showed that under the testing conditions, for Daphnia magna reproductions, the NOEC was not less than the concentration of the 100% saturated solution in the test medium, the LOEC was higher than the concentration of the 100% saturated solution in the test medium, and 50% reduction in reproductive output (EC50) was higher than the concentration of the saturated solution in the test medium. No toxicity was observed under test conditions in the range of water solubility.

 

The measured concentration of the 100% saturated solution was below the limit of detection (LOD) and the LOD was 0.214 mg/L.

Description of key information

The effect of 1,2,4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters on Daphnia magna reproductive output, expressed as The concentration of the 100% saturated solution in the test medium,is as following:

 

The 21d NOEC was not less than the concentration of the saturation solution in the test medium (the measured concentration of the 100% saturated solution was below the limit of detection (LOD) and the LOD was 0.214mg/L). No toxicity was observed under test conditions in the range of water solubility.;

 

The 21d LOEC was higher than the concentration of the saturation solution in the test medium (the measured concentration of the 100 % saturated solution was below the limit of detection (LOD) and the LOD was 0.214 mg/L);

 

The 21d EC50 (50% reduction of the reproductive output) was higher than the concentration of the saturation solution in the test medium (the measured concentration of the 100% saturated solution was below the limit of detection (LOD) and the LOD was 0.214mg/L).  

 

The mortality of the parent animals in control group was 0%, not exceed 20%, and the mortality of the parent animals in the treatment group was 0% at the end of the test.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

>= 100 mg/L