Bis(2-ethylhexyl) succinate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 March 2013 - 15 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

At test initiation and test termination (48 hours), duplicate 100 mL samples were removed from each test solution and control and analysed for the test material. Samples analysed at 0 hours were removed from the test solutions in the volumetric flasks prior to filling the individual test flasks. Samples analysed at 48 hours were removed from composite solutions of replicate vessels for each treatment and the controls.
Storage stability samples were prepared at 1.0, 16 and 100 mg/L, stored and analysed along with the exposure solutions to identify if there were any effects during the transportation of the exposure solutions.

Vehicle:

no

Details on test solutions:

Exposure solutions were prepared individually. The test material was weighed out, transferred to a 2000 mL volumetric flask and made to volume with test media. Test solutions were prepared at nominal concentrations of 1.0, 2.5, 6.3, 16, 40 and 100 mg/L.
The solutions were left to stir overnight for approximately 20 hours. On the day of testing, the stirring was stopped and the solutions were left to stand for approximately 4 hours. The exposure solutions were transferred to an intermediate glass beaker using a syphon from the middle of the flasks to avoid transferring any settled test material. The solutions appeared clear at all concentrations; however at the time of organism addition (0 hours), an oily substance was noted on the surface of the aquatic media in the top two dose levels.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Cultures: DAP 13/42, 43 and 44
- Source: obtained from laboratory cultures maintained at the test laboratory
- Age at study initiation: <24 hours old. On the day of study initiation, adult Daphnia magna were isolated from cultures. Offspring produced from these adults were impartially selected and added to the test vessels.
- Method of breeding: Daphnids were cultured in Elendt M4 media
- Feeding during test: No

ACCLIMATION
- Acclimation conditions (same as test or not): Yes. Daphnia were cultured in media identical to that used in the toxicity test. The daphnid culture area received a regulated photoperiod of 16 hours of light and 8 hours of darkness.
- Type and amount of food: Unicellular green algae, Ankistrodesmus falcatus (4 x 10⁷ cells/mL), at a rate of 1.0 to 2.0 mL per vessel per day based on daphnid age.
- Feeding frequency: Daily

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

224 mg/L

Test temperature:

19.3 - 20.7 °C

pH:

7.22 - 7.40 at test initiation
8.17 - 8.37 at test termination

Dissolved oxygen:

9.98 - 10.84 mg/L at test initiation
10.09 - 10.39 mg/L at test termination

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal test concentrations of 1.0, 2.5, 6.3, 16, 40 and 100 mg/L.

At 0 hours the measured concentrations ranged from 0.0811 to 2.86 mg/L. At 48 hours the measured concentrations above the limit of detection (LOD), assessed as 0.035 mg/L, were not detected. Following this, all results are based on geometric mean measured concentrations.

Geometric mean concentrations: 0.053, 0.087, 0.148, 0.134, 0.203 and 0.316 mg/L for the nominal dose levels of 1.0, 2.5, 6.3, 16, 40 and 100 mg/L, respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass beakers each containing 200 mL of test solution. The test vessels were impartially placed in a temperature-controlled incubator designed to maintain exposure solution temperatures at 20 ± 1 °C.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 media was used during this study to prepare the final exposure solutions. The media was prepared by adding appropriate amounts of sterile nutrient stock solutions to demineralised water.
- Total organic carbon: <3 mg/L
- Conductivity: 0.54 mS/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH, temperature and dissolved oxygen concentration were measured at 0 and 48 hours. Measurements at test initiation were conducted on the test solution remaining after the individual test flasks had been filled. At test termination, after biological observations were completed, the replicate solutions for each treatment and the control were respectively composited for water quality measurements.
pH was measured with a portable pH meter and the dissolved oxygen measured with a VWR pHenomenal OXY 4000H dissolved oxygen meter.
In addition, continuous temperature monitoring was performed inside the test incubator using a surrogate vessel containing demineralised water and a Minimum-Maximum thermometer.

OTHER TEST CONDITIONS
- Adjustment of pH: The final media was adjusted to pH 7.8 ± 0.2.
- Photoperiod: 16 hours light, 8 hours dark (same as in the culture area)
- Light intensity: Not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
The number of immobilised daphnids in each replicate test vessel was recorded at 24 and 48 hours of exposure. Immobilisation was defined as those animals not able to swim within 15 seconds after gentle agitation of the test vessel. Biological observations were also made and recorded at 0, 24 and 48 hours.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Range finding study: Yes
- Test concentrations in the range-finder: Daphnids were exposed, under static conditions, to nominal concentrations of 0.1, 1.0, 10 and 100 mg/L and a control.
- Results used to determine the conditions for the definitive study: Yes. Two test vessels, each containing five daphnids, were established for each treatment level and control. Following 48 hours of exposure, immobilisation of 0, 0, 10 and 10 out of 10 was observed among daphnids exposed to the 0.1, 1.0, 10 and 100 mg/L treatment rates, respectively. Based on these results, an initial experiment was conducted using nominal test concentrations of 0.56, 1.0, 1.8, 3.2, 5.6 and 10 mg/L. Insufficient immobilisation was observed at the highest test concentration during this test to enable an estimation of the 48-hour EC50 value therefore the test concentration for the definitive test was modified. Nominal concentrations of 1.0, 2.5, 6.3, 16, 40 and 100 mg/L were therefore selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.134 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Given that the mechanism of toxicity is considered due to the physical properties and characteristics of the test material, it can be concluded that a true 48 hr-EC50 value could not be determined but can be considered to be > 0.134 mg/L.

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.134 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Given that the mechanism of toxicity is consiered due to the physical properties and characteristics of the test material, it can be concluded that the NOEC for mortality observed in this study was 0.134 mg/L.

Details on results:

BIOLOGICAL RESULTS
Following 48 hours of exposure, immobilisation of 0, 5, 15, 0, 95 and 100 % was observed among daphnids exposed to the 0.053, 0.087, 0.148, 0.134, 0.203 and 0.316 mg/L treatment levels, respectively. All immobilised daphnids were observed to be trapped at the surface. No adverse effects were observed in the surviving daphnia exposed to treatment levels below 0.134 mg/L. All biological observations are presented in Tables 3 and 4.

EVALUATION OF TEST CONDITIONS
Throughout the exposure period, all water quality parameters measured remained within acceptable ranges for the survival of daphnids.

ANALYTICAL MEASUREMENTS
At 0 hours the measured concentrations ranged from 0.0811 to 2.86 mg/L. At 48 hours the measured concentrations above the limit of detection (LOD), assessed as 0.035 mg/L, were not detected. Given the results from the 48-hour analyses, all results are based on geometric mean measured concentrations.

DISCUSSION
The observation of oily substance on the surface of the top two test concentrations (0.203 and 0.316 mg/L as geometric mean measured concentrations) indicates that the test material was present in an undissolved/undispersed form despite attempts to only transfer dissolved/dispersed test material in media for dosing. In addition, sub-lethal observations noted in those daphnia immobilised at 0.203 and 0.316 mg/L by 48 hours corresponded to a physical effect (trapped in meniscus/ at surface).
Sub-lethal observations of being trapped in the meniscus/surface were noted at lower concentrations (0.134 and 0.148 mg/L as geometric mean measured concentrations). This observation was generally reversible, with a very marked decrease of trapped daphnia observed at these rates at 48 hours. Nevertheless this effect is considered to be due to a physical effect of the test material.
Furthermore, as trapped daphnia observations fell between 24 and 48 hours, a low number of daphnids treated at 0.087 mg/L were noted to have attached debris which further demonstrates the likely physical mechanism of the toxicity of the test material to Daphnia magna.

Results with reference substance (positive control):

A reference mortality study using potassium dichromate was performed on D. magna at the test laboratory several months prior to this study. During this study, daphnids were exposed to 0.1, 0.2, 0.36, 0.65 and 1.2 mg/L of potassium dichromate for 48 hours.
After the 48 hour exposure, immobilisations of 5, 95 and 100 % was observed among the D. magna exposed to the 0.36, 0.65 and 1.20 mg/L treatment levels, respectively. Based on these results, the 24 and 48-hour EC50 values were calculated as 0.78 and 0.48 mg/L with 95 % confidence intervals of 0.70 to 0.87 mg/L and 0.45 to 0.53 mg/L, respectively. This result is within the expected range.

Table 3: Concentrations tested, corresponding cumulative percent and number of immobilised organisms during the 48-hour static exposure of daphnids (Daphnia magna) to the test material

Test Concentration (mg/L)	Cumulative Percent Immobilisation (%)*
	24 Hours (Replicates 1 to 4)					48 Hours (Replicates 1 to 4)
	1	2	3	4	Mean	1	2	3	4	Mean
Control	0 (0)	0 (0)	0 (0)	0 (0)	0	0 (0)	0 (0)	0 (0)	0 (0)	0
0.053	0 (0)	0 (0)	0 (0)	0 (0)	0	0 (0)	0 (0)	0 (0)	0 (0)	0
0.087	0 (0)	0 (0)	0 (0)	0 (0)	0	20 (1)	0 (0)	0 (0)	0 (0)	5
0.148	0 (0)	0 (0)	0 (0)	0 (0)	0	20 (1)	0 (0)	20 (1)	20 (1)	15
0.134	0 (0)	0 (0)	0 (0)	20 (1)	5	0 (0)	0 (0)	0 (0)	0 (0)	0
0.203	100 (5)	100 (5)	100 (5)	100 (5)	100	100 (5)	100 (5)	80 (4)	100 (5)	95
0.316	100 (5)	100 (5)	100 (5)	100 (5)	100	100 (5)	100 (5)	100 (5)	100 (5)	100

*Actual number of immobilised daphnids is presented in parentheses

 

Table 4: Concentrations tested and observations made during the 48-hour static exposure of daphnids (Daphnia magna) to the test material

Test Concentration (mg/L)	Observations
	24 Hours (Replicates 1 to 4)				48 Hours (Replicates 1 to 4)
	1	2	3	4	1	2	3	4
Control	5N	5N	5N	5N	5N	5N	5N	5N
0.053	5N	5N	5N	5N	5N	5N	5N	5N
0.087	2N 3T	3N 2T	3N 2T	2N 3T	3N 1D	5N	5N	4N 1D
0.148	5T	5T	5T	5T	3T 1N	5N	4N	3N 1T
0.134	4MS/T 1T	3MS/T 2N	2MS/T 3T	4MS/T	5T	3MS/T 2N	2MS/T 2T 1N	2T 3N
0.203	-	-	-	-	-	-	1T	-
0.316	-	-	-	-	-	-	-	-

N = Normal behaviour

T =Daphnia trapped in meniscus

D = Debris attached to daphnia

MS =Daphnia observed to have reduced movement compared to controls

- = No surviving daphnia

Validity criteria fulfilled:

yes

Conclusions:

Given that the mechanism of toxicity to Daphnia magna was due to the physical properties and characteristics of the test material it can be concluded that the NOEC for mortality observed in this study was 0.134 mg/L and that a true 48 hour EC50 value could not be determined but is considered to be > 0.134 mg/L.

Executive summary:

The short term toxicity of the test material to Daphnia magna was investigated in a study conducted in accordance with the standardised guideline OECD 202 under GLP conditions.

Daphnia were exposed to the test material at nominal concentrations of the test material at 1.0, 2.5, 6.3, 16, 40 and 100 mg/L for 48 hours under static conditions. A control group was exposed to the M4 medium dilution water under the same conditions.

This was a limit of solubility test as the material was not soluble in water. The nominal solutions were prepared and stirred overnight and allowed to stand for approximately 4 hours. The solution used in the test was transferred using a syphon to avoid the transference of any settled test material.

At 0 hours the measured concentrations ranged from 0.0811 to 2.86 mg/L. At 48 hours the measured concentrations above the limit of detection (LOD), assessed as 0.035 mg/L, were not detected. Given the results from the 48-hour analyses, all results are based on geometric mean measured concentrations.

The geometric mean concentrations achieved were 0.053, 0.087, 0.148, 0.134, 0.203 and 0.316 mg/L for the nominal dose levels of 1.0, 2.5, 6.3, 16, 40 and 100 mg/L, respectively.

Following 48 hours of exposure, immobilisation of 0, 5, 15, 0, 95 and 100 % was observed among daphnids exposed to the 0.053, 0.087, 0.148, 0.134, 0.203 and 0.316 mg/L treatment levels, respectively. All immobilised daphnids were observed to be trapped at the surface. No adverse effects were observed in the surviving daphnia exposed to treatment levels below 0.134 mg/L.

An observation of oily substance on the surface of the top two test concentrations (0.203 and 0.316 mg/L as geometric mean measured concentrations) indicates that the test material was present in an undissolved/undispersed form despite attempts to only transfer dissolved/dispersed test material in media for dosing. In addition, sub-lethal observations noted in those daphnia immobilised at 0.203 and 0.316 mg/L by 48 hours corresponded to a physical effect (trapped in meniscus/ at surface).

Sub-lethal observations of being trapped in the meniscus/surface were noted at lower concentrations (0.134 and 0.148 mg/L as geometric mean measured concentrations). This observation was generally reversible, with a very marked decrease of trapped daphnia observed at these rates at 48 hours. Nevertheless this effect is considered to be due to a physical effect of the test material.

Furthermore, as trapped daphnia observations fell between 24 and 48 hours, a low number of daphnids treated at 0.087 mg/L were noted to have attached debris which further demonstrates the likely physical mechanism of the toxicity of the test material to Daphnia magna.

Given that the mechanism of toxicity to Daphnia magna was considered to be due to the physical properties and characteristics of the test material it can be concluded that the NOEC for mortality observed in this study was 0.134 mg/L. A true 48 hour EC50 value could not be determined but is considered to be > 0.134 mg/L.

Description of key information

Study conducted to recognised testing guideline with GLP certification.

Additional information

The short term toxicity of the test material to Daphnia magna was investigated in a study conducted in accordance with the standardised guideline OECD 202 under GLP conditions.

Daphnia were exposed to the test material at nominal concentrations of the test material at 1.0, 2.5, 6.3, 16, 40 and 100 mg/L for 48 hours under static conditions. A control group was exposed to the M4 medium dilution water under the same conditions.

This was a limit of solubility test as the material was not soluble in water. The nominal solutions were prepared and stirred overnight and allowed to stand for approximately 4 hours. The solution used in the test was transferred using a syphon to avoid the transference of any settled test material.

At 0 hours the measured concentrations ranged from 0.0811 to 2.86 mg/L. At 48 hours the measured concentrations above the limit of detection (LOD), assessed as 0.035 mg/L, were not detected. Given the results from the 48-hour analyses, all results are based on geometric mean measured concentrations.

The geometric mean concentrations achieved were 0.053, 0.087, 0.148, 0.134, 0.203 and 0.316 mg/L for the nominal dose levels of 1.0, 2.5, 6.3, 16, 40 and 100 mg/L, respectively.

Following 48 hours of exposure, immobilisation of 0, 5, 15, 0, 95 and 100 % was observed among daphnids exposed to the 0.053, 0.087, 0.148, 0.134, 0.203 and 0.316 mg/L treatment levels, respectively. All immobilised daphnids were observed to be trapped at the surface. No adverse effects were observed in the surviving daphnia exposed to treatment levels below 0.134 mg/L.

An observation of oily substance on the surface of the top two test concentrations (0.203 and 0.316 mg/L as geometric mean measured concentrations) indicates that the test material was present in an undissolved/undispersed form despite attempts to only transfer dissolved/dispersed test material in media for dosing. In addition, sub-lethal observations noted in those daphnia immobilised at 0.203 and 0.316 mg/L by 48 hours corresponded to a physical effect (trapped in meniscus/ at surface).

Sub-lethal observations of being trapped in the meniscus/surface were noted at lower concentrations (0.134 and 0.148 mg/L as geometric mean measured concentrations). This observation was generally reversible, with a very marked decrease of trapped daphnia observed at these rates at 48 hours. Nevertheless this effect is considered to be due to a physical effect of the test material.

Furthermore, as trapped daphnia observations fell between 24 and 48 hours, a low number of daphnids treated at 0.087 mg/L were noted to have attached debris which further demonstrates the likely physical mechanism of the toxicity of the test material to Daphnia magna.

Given that the mechanism of toxicity to Daphnia magna was considered due to the physical properties and characteristics of the test material it can be concluded that the NOEC for mortality observed in this study was 0.134 mg/L. A true 48 hour EC50 value could not be determined but is considered to be > 0.134 mg/L.

The study was conducted to GLP and a standardised guideline. It was therefore assigned a reliability score of 1 and considered suitable for assessment as an accurate reflection of the test substance.