Reaction mass of N-butylphthalimide and N-sec-butylphthalimide and N-propylphthalimide

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:

9 December 2016 - 5 January 2017

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)

Version / remarks:

13 April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch No: M1605-05371-01
Storage: At room temperature, ca. 20°C

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The following test vessels were set up:
- Test solution (Tn); containing Daphnia medium with test item (two replicates with 20 Daphnia each)
- Blank control (Bn); containing pure Daphnia medium (three replicates with 20 Daphnia each)
- Since the test item is a mixture with a limited solubility, only the water-soluble fractions (WSFs)3 were tested. These WSFs were prepared, separately for each test concentration, by adding the respective quantities of test item to empty glass vessels (using a Hamilton pipette, considering a density of 1.12 g/ml), adding the Daphnia medium, and stirring these mixtures for 96 h to maximize dissolution. Subsequently, the WSFs were left standing for about an hour to allow for separation of the test item from the water phase. The aqueous phase was then sampled from the middle of the vessel with a long tube, as a small accumulation of test item was visible in the outlet at the bottom. The resulting solutions were used as test solutions in the toxicity test. Pure Daphnia medium served as blank controls.
- O2 concentration and pH were measured in the test medium for each concentration including the blank control. If the O2 concentration was too low (>5.0 mg/l required) the media was aerated by stirring. Afterwards, the media were filled into the test vessels (50 ml per vessel).
- Daphnia, aged less than 24 h and already acclimatized to the Daphnia medium, were then introduced into the test media, and the vessels were covered with a glass plate. The Daphnia were not fed during the test and the test vessels were not aerated.
Static conditions: duration 48 h
- Evidence of undissolved material: Since the test item is a mixture with a limited solubility, only the water-soluble fractions (WSFs)3 were tested

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISMS
- Test organism: Daphnia magna (Straus, 1820), derived from a healthy stock and not first brood progeny
- Breeding: Parental and young Daphnia held in 4 l glass aquaria (3 l medium) at 20±2°C
- Illumination: 16 h per day
- Medium: Continuously aerated Elendt M4 medium prepared with ultra-pure water (conductivity <1.5 µS/cm)
- Feed: Suspension of Desmodesmus subspicatus in Elendt M4 medium with an optical density OD680 of about 15 units

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

not reported

Test temperature:

18–22°C, controlled at ±1 °C

pH:

6.6 to 8.1

Dissolved oxygen:

The minimum dissolved oxygen concentration in the controls and the test vessels at the end of each 24h-period was >=7.0 mg O2/l (required >=3 mg O2/l).

Salinity:

not reported

Conductivity:

497 to 768 µS cm-1

Nominal and measured concentrations:

The concentrations of the three main components of the test item in the test media were determined by HPLC analysis at the beginning of the test and after 24 and 48 h of exposure. These analyses confirmed their limited and different solubilities, and showed that they all were decreasing over the whole 48-h test period. While the sum of the three components accounted for 51, 41, 39, 20 and 18% of the respectively 100, 50.0, 25.0, 12.5 and 6.25 mg/l loadings at the beginning of the test, it dropped down to 36, 29, 25, 12 and 14% after 48 hours of exposure.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml beakers, all-glass, with 50 ml of test medium,covered with a glass plate to avoid evaporation and contamination of the test solutions with dust
- Test medium: Elendt M4 medium; prepared with ultra-pure water (conductivity <1.5 µS/cm)
- Number of Daphnia: xx individuals per test concentration, xx per vessel (check!)
- Age: Less than 24 h
- Light: 16 h photoperiod a day, supplied by overhead white fluorescent tubes
- Temperature: 18–22 °C, controlled at ±1 °C
- pH: 6 to 9. The pH should normally not vary by more than 1.5units in one test.
- Feed: The Daphnia are not fed during the test
- Aeration: The test vessels were not aerated during the test.
- Test type: Static exposure conditions
- Test duration: 48 h

RANGE-FINDING STUDY
Prior to the definitive test a non-GLP range finding test with loading rates of 1, 10 and 100 mg/l of Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide was performed. The concentrations of the three main test item components were determined with HPLC in the test solutions at the beginning and at the end of the test. These measurements showed that the three main test item components were only dissolved to about 70% of the nominal loading, and that the determined test concentrations remained stable over the 48 h test period (>=80% of the initial value). Therefore, static conditions can be applied in the definitive test.

EFFECT PARAMETERS MEASURED
Observed immobility (inability to swim) of the daphnids: Observations of immobile Daphnia were made after 24 and 48 h of exposure. Any abnormal behaviour or appearance was reported.

Reference substance (positive control):

yes

Remarks:

Acute reference test with potassium dichromate conducted twice a year

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

1.24 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

4.64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.979 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

1.82 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

9.21 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

1.05 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

After 24 h, following immobilization rates were observed: 92.5% at 100 mg/l, 62.5% at 50.0 mg/l, 55% at 25.0 mg/l and 17.5% at 12.5 mg/l. No significant effects (<=10% immobilization) were observed at 6.25 mg/l or in the blank controls.
Based on these effects and the measured concentrations of the test item, the 24h-EC50 of Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide to Daphnia magna was calculated to be 9.21 mg/l (95% confidence limits: 6.99–12.2 mg/l). The low effect concentration EC10 after 24h was calculated to be 1.82 mg/l (95% confidence limits: 1.05–2.67 mg/l).
The no observed effect concentration (NOEC) after 24h was determined to be 1.05 mg/l.

After 48 h, following immobilization rates were observed: 100% at 100 mg/l, 82.5% at 50.0 mg/l, 75% at 25.0 mg/l and 27.5% at 12.5 mg/l. No significant effects (<=10% immobilization) were observed at 6.25 mg/l or in the blank controls.
Based on these effects and the measured concentrations of the test item, the 48h-EC50 of Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide to Daphnia magna was calculated to be 4.64 mg/l (95% confidence limits: 1.98–10.1 mg/l). The low effect concentration EC10 after 48h was calculated to be 1.24 mg/l (95% confidence limits: 0.143–2.24 mg/l).
The no observed effect concentration (NOEC) after 48h was determined to be 0.979 mg/l.

Results with reference substance (positive control):

Acute ref. test with K2Cr2O7 conducted twice a year. The EC50 value for the control of sensitivity for 24 h of exposure with K2Cr2O7 was estimated to be 0.89 mg/l (29.6.2016), which lies within the recomm. range of 0.6–2.1 mg/l acc. to OECD Guideline 202.

Reported statistics and error estimates:

The effective concentrations ECx were assessed based on the geometric mean of the measured concentrations of the sum of the three main components over the whole 48 h test period.
Statistical analysis was performed with respect to ECx values and their 95% confidence intervals whenever possible.

Validity criteria fulfilled:

yes

Remarks:

All validity criteria i.e. immobilization in the controls <=10% and O2 concentrations at the end of the test >=3 mg/L were fulfilled.

Conclusions:

The acute toxicity of reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide to Daphnia magna was determined in a 48 hour static test according to OECD guideline 202. The median effect concentrations (EC50) of reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide on Daphnia magna were estimated to be 9.21 mg/l and 4.64 mg/l measured concentration after 24 and 48 h of exposure, respectively. The NOEC values were determined to be 1.05 mg/l and 0.979 mg/l measured concentration after 24 and 48 h of exposure, respectively. The results of the test can be considered reliable without restriction.

Executive summary:

The median effect concentration (EC50) and the no-effect concentration (NOEC) of reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide to Daphnia magna were investigated under static exposure conditions over a period of 48 h, following guideline OECD 202.

The test item is liquid, poorly soluble and consists of a reaction mass of three components, which account for a total purity of 99.8%. Consequently, the test solutions were prepared as 96-h WSFs (water soluble fractions), separately for each test concentration.

The loading rates (test concentrations) were 100, 50.0, 25.0, 12.5 and 6.25 mg/l.

40 individual Daphnia divided into 2 test vessels were exposed to each concentration of the test item, and 60 Daphnia divided into 3 test vessels were used as blank controls.

The concentrations of the three main components of the test item in the test media were determined by HPLC analysis at the beginning of the test and after 24 and 48 h of exposure. These analyses confirmed their limited and different solubilities, and showed that they all were decreasing over the whole 48-h test period. Therefore, the effective concentrations ECx were assessed based on the geometric mean of the measured concentrations of the sum of the three main components over the whole 48 h test period.

After 24 h, following immobilization rates were observed: 92.5% at 100 mg/l, 62.5% at 50.0 mg/l, 55% at 25.0 mg/l and 17.5% at 12.5 mg/l. No significant effects (<=10% immobilization) were observed at 6.25 mg/l or in the blank controls. After 48 h, following immobilization rates were observed: 100% at 100 mg/l, 82.5% at 50.0 mg/l, 75% at 25.0 mg/l and 27.5% at 12.5 mg/l. No significant effects (<=10% immobilization) were observed at 6.25 mg/l or in the blank controls.

The results of the toxicity of reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide to Daphnia magna after 24 h and 48 h of exposure are summarized in the following table, showing EC-values and 95% confidence limits (cl) based on the measured concentrations of the sum of the three main components:

Parameter, expressed as Measured concentration (mg/l) 24 h of exposure	EC10	EC50	NOEC (1)
Estimate	1.82	9.21	1.05
Lower 95%-cl	1.05	6.99
Upper 95%-cl	2.67	12.2
Parameter, expressed as Measured concentration (mg/l)  48 h of exposure
Estimate	1.24	4.64	0.979
Lower 95%-cl	2.64	10.1
Upper 95%-cl

cl: confidence limit

(1) : no statistical analysis; NOEC = highest concentration for which <=10% immobilization is observed

The 48 h EC50 value of Reaction mass of N-butylphthalimide and N-propylphthalimide and N-secbutylphthalimide to Daphnia magna was 4.64 mg/l. This value is based on measured concentrations. All validity criteria were fulfilled.

Description of key information

The acute toxicity of reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide to Daphnia magna was determined in a 48 hour static test according to OECD guideline 202. The median effect concentrations (EC50) of reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide on Daphnia magna were estimated to be 9.21 mg/l and 4.64 mg/l measured concentration after 24 and 48 h of exposure, respectively. The NOEC values were determined to be 1.05 mg/l and 0.979 mg/l measured concentration after 24 and 48 h of exposure, respectively. The results of the test can be considered reliable without restriction.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

4.64 mg/L