Benzene-1,2,4-tricarboxylic acid 1,2-anhydride, oligomeric reaction products with ethane-1,2-diol and glycerol

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:

This study was conducted between 21 July 2017 and 28 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Version / remarks:

EC No. 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Information as provided by the Sponsor.
Identification: Benzene-1,2,4-tricarboxylic acid 1,2-anhydride, oligomeric reaction products with ethane-1,2-diol and glycerol
Batch: AAE1455700
Purity: not supplied
Appearance: beige solid flakes
Expiry Date: 10 October 2018
Storage Conditions: room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

Range-Finding Test
A sample of each test concentration was taken for chemical analysis on Days 0 and 3 in order to determine the stability of the test item under test conditions, however, no analysis of the samples was conducted

Definitive Test
The concentration of measured carbon within the test preparations was verified by TOC analysis on Days 0, 2, 5, 7, 9, 12, 14, 16, 19 and 21

Vehicle:

no

Details on test solutions:

Range-finding Test
The test concentrations to be used in the definitive test were determined by a preliminary range finding test.
Nominal amounts of test item (20 and 200 mg) were separately added to the surface of 2 liters of test water to give the 10 and 100 mg/L loading rates respectively. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1-Hour. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid depth siphoning (the first approximate 75 to 100 mL discarded) to give the 10 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no micro dispersions or undissolved test item to be present.

Definitive Test
Based on the results of a preliminary range finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to a WAF of the test item over a range of test concentrations of 5.6, 10, 18, 32, 56, and 100 mg/L for a period of 21 days. The test solutions were renewed 3 times per week.
Prior to addition of the test item a glass siphon tube was placed in the test media. Nominal amounts of test item (11.2, 20, 36, 64, 112 and 200 mg) were each separately added to the surface of 2 liters of test water to give the 5.6, 10, 18, 32, 56 and 100 mg/L loading rates respectively. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1-Hour. Visual observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2 to 4 cm in length). A length of Tygon tubing was attached to the top of the glass siphon tube. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid depth siphoning (the first approximate 75 to 100 mL discarded) to give the 5.6, 10, 18, 32, 56 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no micro dispersions or undissolved test item to be present

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium (see Annex 1) in a temperature controlled room at approximately 20 C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

static

Water media type:

freshwater

Remarks:

The reconstituted water (Elendt M7 medium) used for the range finding and definitive tests was the same as that used to maintain the stock animals

Limit test:

no

Total exposure duration:

21 d

Hardness:

240 - 280 mg/L as CaCO3

Test temperature:

21 - 22 ”C

Dissolved oxygen:

8.3 - 9.0 mg O2/L

Details on test conditions:

Experimental Design and Study Conduct
Validation of Mixing Period
Preliminary work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, as an indicator of soluble organic substances.

Range-finding Test
The test concentrations to be used in the definitive test were determined by a preliminary range finding test.
In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 10 and 100 mg/L.
Nominal amounts of test item (20 and 200 mg) were separately added to the surface of 2 liters of test water to give the 10 and 100 mg/L loading rates respectively. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1-Hour. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid depth siphoning (the first approximate 75 to 100 mL discarded) to give the 10 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no micro dispersions or undissolved test item to be present.
In the range finding test, for each concentration a single daphnid was placed in 100 mL of the test preparation in 150 mL glass vessels which were then covered with a plastic lid to reduce evaporation. For each test and control group 5 replicate test vessels were prepared. The water temperature was maintained at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for 10 days.
The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were renewed on Days 0, 3, 5 and 7. The adult daphnia were transferred to fresh media by wide-bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted using a stereo microscope before being discarded.
Each daphnid received approximately 5 or 15 µL of an algal suspension (Desmodesmus subspicatus) and approximately 20 µL of Tetramin® flake food suspension daily. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.
A sample of each test concentration was taken for chemical analysis on Days 0 and 3 in order to determine the stability of the test item under test conditions, however, no analysis of the samples was conducted.

Definitive Test
Based on the results of a preliminary range finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to a WAF of the test item over a range of test concentrations of 5.6, 10, 18, 32, 56, and 100 mg/L for a period of 21 days. The test solutions were renewed 3 times per week.
Prior to addition of the test item a glass siphon tube was placed in the test media. Nominal amounts of test item (11.2, 20, 36, 64, 112 and 200 mg) were each separately added to the surface of 2 liters of test water to give the 5.6, 10, 18, 32, 56 and 100 mg/L loading rates respectively. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1-Hour. Visual observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2 to 4 cm in length). A length of Tygon tubing was attached to the top of the glass siphon tube. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid depth siphoning (the first approximate 75 to 100 mL discarded) to give the 5.6, 10, 18, 32, 56 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no micro dispersions or undissolved test item to be present.
The concentration of measured carbon within the test preparations was verified by TOC analysis on Days 0, 2, 5, 7, 9, 12, 14, 16, 19 and 21 (see Annex 3).

Exposure Conditions
For each concentration a single daphnid was placed in 100 mL of the test preparation in 150 mL glass vessels which were then covered with a plastic lid to reduce evaporation. For each test and control group 10 replicate test vessels were prepared. The test vessels were maintained in a temperature controlled room at 18 to 22 C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light (not exceeding 1500 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days. The test vessels were not aerated. The diluent water only was aerated prior to use.
The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were renewed 3 times per week on Days 0, 2, 5, 7, 9, 12, 14, 16 and 19. The adult daphnia were transferred to fresh media by wide bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted using a stereo microscope before being discarded.
Each daphnid received approximately 5 or 15 µL of an algal suspension (Desmodesmus subspicatus) and approximately 20 µL of Tetramin® flake food suspension daily. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

58 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

65 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth

Remarks:

body length

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth

Remarks:

body length

Key result

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

> 56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth

Remarks:

body length

Details on results:

Range-finding Test
Cumulative immobilization and sub lethal effects data from the exposure of Daphnia magna to the test item during the range finding test are given in Table 1.
No immobilization was observed at the test concentration of 10 mg/L Loading Rate WAF, however, 100% immobilization was observed at the test concentration of 100 mg/L Loading Rate WAF.
Analysis of the range-finding test preparations was not conducted.
Based on this information test concentrations of 5.6, 10, 18, 32, 56 and 100 mg/L were selected for the definitive test.

Definitive Test
Based on the results of a preliminary range finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to a WAF of the test item over a range of test concentrations of 5.6, 10, 18, 32, 56 and 100 mg/L for a period of 21 days. The test solutions were renewed 3 times per week.

Total Organic Carbon Analysis
TOC analysis of the fresh test preparations on Days 0, 5, 9, 14 and 19 showed measured carbon concentrations to range from 0.18 to 56 mg/L. TOC analysis of the expired test preparations on Days 2, 7, 12, 16 and 21 showed measured carbon concentrations to range from less than the TOC in the control to 57 mg/L.
Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Test Organism Observations
The observations for each test and control group are summarized in Table 3. The total cumulative production of live young is given in Table 4 and the number of live young produced per adult is shown in Table 2.

Lethal Effects on the Parental Generation (P1)
Mortality (immobilization) occurred predominantly at the highest test loading rate of 100 mg/L resulting in 100% mortality by Day 9. Mortality was also observed at the test loading rates of 5.6, 10, 32 and 56 mg/L, however, statistical analysis of the mortality data using the Step-down Cochran-Armitage Test procedure showed that the observed mortalities in the 5.6, 10, 32 and 56 mg/L loading rate WAF groups were not significantly different (P0.05) when compared to the control group.
No mortalities occurred at 18 mg/L loading rate WAF throughout the test.
The following ELx (immobilization) values based on nominal loading rates were calculated by the Probit analysis using Linear Maximum Likelihood regression at 21 days
Endpoint Concentration (mg/L Loading Rate WAF)
Immobilization EL10 33
95% confidence limits Not determined
EL50 58
95% confidence limits Not determined
No Observed Effect Loading Rate 56
Lowest Observed Effect Loading Rate 100

Sub-lethal Effects on the Parental Generation (P1)
After 21 days the length of each surviving adult was determined, the results of which are given in Table 5.
The results showed that there were no statistically significant differences (P>0.05) between the control and the 5.6, 10, 18, 32 and 56 mg/L loading rate WAF test groups in terms of length of the daphnids after 21 days exposure to the test item. The 100 mg/L loading rate WAF group was not included in this analysis as exposure to the test item eliminated all the daphnids prior to Day 21 of the test.
The following ELx (parental body length) values based on nominal loading rates were estimated by inspection of the data at 21 days:
Endpoint Concentration (mg/L Loading Rate WAF)
Parental Body Length EL10 >56
95% confidence limits Not determined
EL50 >56
95% confidence limits Not determined
No Observed Effect Loading Rate 56
Lowest Observed Effect Loading Rate >56

Effects on Reproduction
After 21 days there were no statistically significant differences between the control and the 5.6, 10, 18, 32 and 56 mg/L loading rate WAF groups in terms of the number of live young produced per adult. The 100 mg/L loading rate WAF group showed a statistically significant difference from the control after 21 days in terms of producing fewer numbers of live young per adult (see Table 4).
The following ELx (reproduction) values based on nominal loading rates were calculated by Logit analysis using Linear Maximum-Likelihood regression at 21 days:
Endpoint Concentration (mg/L Loading Rate WAF)
Reproduction EL10 60
95% confidence limits Not determined
EL50 65
95% confidence limits Not determined
No Observed Effect Loading Rate 56
Lowest Observed Effect Loading Rate 100

Effects on the Filial Generation (F1)
Information on the effects of the test item on the F1 generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch. However, an assessment made at each observation showed the "filial" daphnids produced by the 5.6, 10, 18, 32 and 56 mg/L loading rate WAF groups were in the same general condition as the young produced by the controls over the duration of the test with the following exceptions; 5.6 mg/L loading rate WAF group – the neonates within two broods were small and one brood were pale, 10 mg/L loading rate WAF group – the neonates within one brood was small and pale, 32 mg/L loading rate WAF group – the neonates within two broods were small and two broods were small and pale, 56 mg/L loading rate WAF group – the neonates within one brood were small.
Young were first produced in the control test group on Day 8 of the test.
Due to the toxic effect of the test item the parental generation (P1) of the 100 mg/L loading rate WAF group were eliminated prior to the production of young.
There were no unhatched eggs or dead young recorded in all control and treatment groups throughout the test.

Lowest Observed Effect Loading Rate
The LOEL was 100 mg/L as significant mortalities (immobilization) were observed in the parental generation (P1) and this test group produced significantly fewer live young per adult (P<0.05) in terms of the number of live young produced per adult when compared to the control after 21 days.

No Observed Effect Loading Rate
The NOEL was 56 mg/L as there were no significant mortalities (immobilization) observed in the parental generation (P1) and there were no significant differences (P0.05) in terms of the number of live young produced per adult when compared to the control after 21 days.

Water Quality Criteria
Temperature was maintained at approximately 21”C to 22”C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
The water hardness was observed to be in the range 240 to 280 mg/L as CaCO3 in the control and the highest surviving test group throughout the test.
Throughout the test the light intensity was observed to be in the range 369 to 721 lux.
The water temperature was also recorded in the control vessel every hour using a Testo temperature logger.

Vortex Depth Measurements
The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface.

Observations on Test Item Solubility
Observations on the test media were carried out during the mixing and testing of the WAFs.
At the start of the mixing period the 5.6, 10, 18, 32, 56 and 100 mg/L loading rates were observed to be clear colorless water columns with beige flakes of test item on the bottom of the vessel and an oily layer floating at the surface. After 23 hours stirring and a 1 Hour standing period the 5.6, 10, 18, 32, 56 and 100 mg/L loading rate were observed to be clear colorless water columns with test item dispersed throughout, with larger globules on the bottom of the vessel and a slight oily layer floating at the surface. Microscopic inspection of the WAFs following filtration through a glass wool plug showed no micro dispersions or undissolved test item to be present. After siphoning and for the duration of the test, the 5.6, 10, 18, 32, 56 and 100 mg/L loading rates were observed to be clear, colorless solutions.

  Validation Criteria

The following validation criteria were achieved during the test:

	Required	Actual
Control mortality	£20%	10%
Mean number of live young per surviving adult (control group)	³60 after 21 days	126
Coefficient of variation for control group*	25%	15%
No ephippia produced	0	0
Dissolved oxygen	>3 mg O2/L	≥7.6 mg O2/L
pH (control group)	6 to 9 variation 1.5	7.1 to 8.2 1.0

*=   Based on total number of living offspring per parent animal alive at the end of the test

Table3            Summary of ObservationsExcluding Accidental and Inadvertent Mortalities

Day	Nominal Loading Rate (mg/L)
	Control		5.6		10		18		32		56		100
	Adults Surviving	Live Young	Adults Surviving	Live Young	Adults Surviving	Live Young	Adults Surviving	Live Young	Adults Surviving	Live Young	Adults Surviving	Live Young	Adults Surviving	Live Young
1	10	0	10	0	10	0	10	0	10	0	10	0	10	0
2	10	0	10	0	10	0	10	0	10	0	10	0	10	0
3	10	0	10	0	10	0	10	0	10	0	10	0	8	0
4	10	0	10	0	10	0	10	0	10	0	10	0	3	0
5	10	0	10	0	10	0	10	0	10	0	10	0	3	0
6	10	0	10	0	10	0	10	0	10	0	10	0	1	0
7	10	0	10	0	10	0	10	0	10	0	10	11	1	0
8	10	30	10	0	10	0	10	0	10	0	10	0	1	0
9	10	60	10	89	10	117	10	137	10	63	10	115	0	0
10	10	47	10	39	10	18	10	0	10	56	10	18	0	0
11	10	103	10	71	10	87	10	163	10	90	10	92	0	0
12	10	31	10	26	10	62	10	31	10	77	10	52	0	0
13	10	0	10	0	10	0	10	0	10	0	10	0	0	0
14	10	322	10	240	10	287	10	322	10	199	10	163	0	0
15	9	0	10	35	10	0	10	0	10	112	10	32	0	0
16	9	150	10	94	10	175	10	174	10	82	10	101	0	0
17	9	189	10	103	10	156	10	189	10	95	10	80	0	0
18	9	8	10	17	10	0	10	2	9	0	10	54	0	0
19	9	109	9	137	10	162	10	199	9	136	8	100	0	0
20	9	0	7	0	9	0	10	0	9	0	8	0	0	0
21	9	87	7	69	9	31	10	0	9	0	8	47	0	0
TOTAL	9	1136	7	920	9	1095	10	1217	9	1011	8	865	0	0

Table4          Total Cumulative Production of Live Young

Nominal Loading Rate (mg/L)	Day
	1	2	3	4	5	6	7	8	9	10
Control	0	0	0	0	0	0	0	30	109	156
5.6	0	0	0	0	0	0	0	0	134	173
10	0	0	0	0	0	0	0	0	135	153
18	0	0	0	0	0	0	0	0	137	137
32	0	0	0	0	0	0	0	0	92	148
56	0	0	0	0	0	0	11	11	172	190
100	0	0	0	0	0	0	0	0	0	0

 

Nominal Loading Rate (mg/L)	Day
	11	12	13	14	15	16	17	18	19	20	21
Control	279	310	310	665	665	815	1004	1012	1121	1121	1208
5.6	288	314	314	647	682	809	980	997	1195	1195	1264
10	240	320	320	637	637	812	1006	1006	1168	1168	1199
18	300	331	331	653	653	827	1016	1018	1217	1217	1217
32	259	359	359	617	729	823	953	1054	1211	1211	1211
56	319	390	390	648	680	848	928	982	1082	1082	1170
100	0	0	0	0	0	0	0	0	0	0	0

 

Table5            Body Length of Surviving Adults at Day 21

Nominal Loading Rate (mg/L)	Individual Daphnia Lengths (mm)
	1	2	3	4	5	6	7	8	9	10	Av	SD
Control	4.2	4.0	4.1	-	4.0	4.3	4.2	4.1	4.0	4.3	4.1	0.12
5.6	-	3.9	4.1	4.1	-	3.8	4.2	4.1	3.9	-	4.0	0.15
10	4.2	4.1	4.0	4.0	3.9	4.2	4.2	4.2	4.1	-	4.1	0.11
18	3.8	3.9	4.3	4.3	4.1	4.0	3.9	4.2	4.1	4.1	4.1	0.17
32	4.2	4.1	4.1	4.0	-	4.3	4.0	4.1	4.2	-	4.1	0.10
56	4.1	4.2	3.8	4.3	-	-	4.0	3.8	4.0	4.0	4.0	0.18

Av = Average

SD= Standard Deviation

-=    No measurement made as Daphnia died during the test

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to the test item resulted in significant mortalities at the
100 mg/L loading rate resulting in 100% mortality by Day 9.
The 21 Day EL50 (immobilization) value, based on nominal loading rates, for the parental daphnia generation (P1) was calculated to be 58 mg/L.
The 21 Day EL50 (parental body length) based on nominal loading rates was greater than 56 mg/L.
The 21 Day EL50 (reproduction) based on nominal loading rates was 65 mg/L.
The Lowest Observed Effect Loading Rate and the No Observed Effect Loading Rate based on nominal loading rates were 100 and 56 mg/L respectively.

Executive summary:

A study was performed to assess the chronic toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2012) No 211, "Daphnia magna Reproduction Test" referenced as Method C.20 of Commission Regulation (EC) No. 440/2008.

 Methods

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as Water Accommodated Fractions (WAFs).

Based on the results of a preliminary range‑finding test,Daphnia magna were exposed (10 replicates of a single daphnid per group) to a WAF of the test item over a range of nominal loading rates of 5.6,10,18,32,56 and 100 mg/L for a period of 21 days. The test solutions were renewed 3 times per week. 

The numbers of live and dead adult daphnia and young daphnids (live and dead) were determined daily. The daphnia were fed daily with a mixture of algal suspension and Tetramin^®flake food suspension.

 Results

Total Organic Carbon (TOC) analysis of the fresh test preparations on Days 0, 5, 9, 14 and 19 showed measured carbon concentrations to range from 0.18 to 56 mg/L. TOC analysis of the expired test preparations on Days 2, 7, 12, 16 and 21 showed measured carbon concentrations to range from less than the TOC in the control to 57 mg/L.

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of Daphnia magna to the test item gave the following results based on the nominal test concentrations:

Endpoint		Concentration (mg/L Loading Rate WAF)
Immobilization	EL10	33
	95% confidence limits	Not determined
	EL50	58
	95% confidence limits	Not determined
	No Observed Effect Loading Rate	56
	Lowest Observed Effect Loading Rate	100
Reproduction	EL10	60
	95% confidence limits	Not determined
	EL50	65
	95% confidence limits	Not determined
	No Observed Effect Loading Rate	56
	Lowest Observed Effect Loading Rate	100
Body Length	EL10	>56
	95% confidence limits	Not determined
	EL50	>56
	95% confidence limits	Not determined
	No Observed Effect Loading Rate	56
	Lowest Observed Effect Loading Rate	>56

Description of key information

Benzene-1,2,4-tricarboxylic acid 1,2-anhydride, oligomeric reaction products with ethane-1,2-diol and glycerol was tested in a Daphnia magna reproduction test according to OECD 211

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

56 mg/L