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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Sep 2018 - 11 Apr 2019
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:
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
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Version / remarks:
Method C.20 of Commission Regulation (EC) No. 440/2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: Benzyl 3-isobutyryloxy-1-isopropyl-2,2-dimethylpropyl phthalate
Alternative name: Santicizer® 278
CAS Number: 16883-83-3
Batch: 3057
Purity: 98.652%
Physical State/
Appearance: Pale yellow viscous liquid
Expiry Date: 16 January 2020
Storage Conditions: Room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
Definitive test: Water samples were taken from the control and each surviving test group for immediate quantitative analysis. Samples of the fresh test preparations were taken from the bulk test preparations on Days 0, 7, 14 and 19 and of the expired test preparations (replicates pooled) on Days 2, 16 and 21. Duplicate samples were taken on each occasion and stored frozen for further analysis if necessary.

Water Quality Criteria
Dissolved oxygen concentrations, pH and temperature were recorded before and after each test media renewal. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. Measurements were made on one replicate for each test concentration. The temperature was also measured every hour in one replicate of the control using a Testo temperature logger.

The water hardness, using methods described in Fields and On Site Methods for Analysis of Water (British Standards Institution, 1993), was measured in the test water used in the preparation of each batch of test solutions.
Vehicle:
yes
Remarks:
Dimethylformamide
Details on test solutions:
Test Water: 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.

Slow-Stir Preliminary Solubility Trials and Range-Finding Test
Information provided by the Sponsor indicated that the test item was a pure material with low water solubility (0.0015 mg/l); therefore a preliminary media preparation trial was conducted using the slow-stir saturated solution method of preparation. The 100% v/v saturated solution was prepared at a nominal concentration of 50 mg/L with the aid of a magnetic stirrer for 23 hours, using a stirring rate such that a vortex was formed to give a dimple at the water surface to avoid the potential formation of dispersions or micro-emulsions. After 23 hours the stirring was stopped and the mixture allowed to stand for 1 hour prior to removal of the aqueous phase by siphoning, taking care to avoid any undissolved test item. The measured concentration of the 100% v/v saturated solution was determined to be 0.0070 mg/L.

A range-finding test was conducted using a slow-stir saturated solution method of preparation at nominal concentrations of 1.0, 10 and 100% v/v saturated solution. The 100% v/v saturated solution was prepared at a nominal concentration of 50 mg/L with the aid of a magnetic stirrer for 23 hours, using a stirring rate such that a vortex was formed to give a dimple at the water surface. After 23 hours the stirring was stopped and the mixture allowed to stand for one hour prior to removal of the aqueous phase by siphoning, taking care to avoid any undissolved test item. The measured concentrations of the 1.0, 10 and 100% v/v saturated solution were determined to be 0.0031, 0.019 and 0.21 mg/L, and were therefore considerably different to the measured concentration determined in the preliminary media preparation trial.

As a result of the inconsistent measured concentrations determined in the slow-stir media preparation trial and slow-stir range-finding test, a further media preparation trial was conducted. In this trial triplicate 100% v/v saturated solutions were prepared at a nominal concentration of 50 mg/L with the aid of a magnetic stirrer for 23 hours, using a stirring rate such that a vortex was formed to give a dimple at the water surface. After 23 hours the stirring was stopped and the mixture allowed to stand for one hour prior to removal of the aqueous phase by siphoning, taking care to avoid any undissolved test item. Two samples were taken from each preparation, the first after discarding 1 liter of solution and the second after discarding a further 5 liters. The measured concentrations from this trial (see table below) indicated that the concentrations within a preparation were consistent but that the concentrations between preparations were variable.

Measured Concentration (mg/L)
Sampled after 1 Liter Discarded Sampled after 5 Liters Discarded
Preparation 1 0.0039 0.0042
Preparation 2 0.018 0.019
Preparation 3 0.037 0.040

Given this information, it was concluded that the slow-stir method of preparation was not appropriate for this test item and therefore a solvent spike media preparation trial was conducted.


Solvent Spike Media Preparation Trial:
In the solvent spike media preparation trial, a nominal amount of test item (20 mg) was dissolved in dimethylformamide (DMF) and the volume adjusted to 10 mL to give a 2.0 mg/mL stock solution from which a series of dilutions was made to give further solvent stock solutions of 0.060 and 0.60 mg/mL. The solvent stock solutions were inverted several times to ensure adequate mixing and homogeneity. An aliquot (50 µL) of each solvent stock solution was separately dispersed in 2 liters of test water with the aid of magnetic stirring for approximately 5 minutes to produce the required test concentrations of 0.0015 and 0.015 mg/L in triplicate. Measured concentrations of the 0.015 mg/L preparations ranged from 0.013 to 0.014 mg/L (83% to 95% of the nominal concentration) and in the 0.0015 mg/L preparations ranged from 0.00099 to 0.0012 mg/L (66% to 77% of the nominal concentration). Given this information, it was concluded that the testing should proceed using the solvent spike method of preparation.

Range finder test item preparation
The test concentrations to be used in the definitive test were determined by a solvent spike range finding test. In the range finding test Daphnia magna were exposed to a series of nominal concentrations of 0 (control), 0 (solvent control), 0.0015, 0.0050 and 0.015 mg/L.

A nominal amount of test item (20 mg) was dissolved in dimethylformamide (DMF) and the volume adjusted to 10 mL to give a 2.0 mg/mL stock solution from which a series of dilutions was made to give further solvent stock solutions of 0.060, 0.20 and 0.60 mg/mL. The solvent stock solutions were inverted several times to ensure adequate mixing and homogeneity. An aliquot (50 µL) of each solvent stock solution was separately dispersed in 2 liters of test water with the aid of magnetic stirring for approximately 5 minutes to produce the required test concentrations of 0.0015, 0.0050 and 0.015 mg/L. An error on Day 0 resulted in the 0.060 mg/mL stock solution being prepared at 0.0060 mg/mL, which in turn meant the 0.0015 mg/L test solution was prepared at 0.00015 mg/L. The control and the solvent control groups were maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 50 µl/L of dimethylformamide.

Definitive test test item preparation
Based on the results of a solvent spike range finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to solutions of the test item at nominal concentrations of 0 (control), 0 (solvent control), 0.0015, 0.0050 and 0.010 mg/L for a period of 21 days. The test solutions were renewed three times per week.

A nominal amount of test item (20 mg) was dissolved in dimethylformamide (DMF) and the volume adjusted to 10 mL to give a 2.0 mg/mL stock solution from which a series of dilutions was made to give further stock solutions of 0.060, 0.20 and 0.40 mg/mL. The stock solutions were inverted several times to ensure adequate mixing and homogeneity. An aliquot (50 µL) of each solvent stock solution was separately dispersed in 2 liters of test water, with the aid of magnetic stirring for approximately 5 minutes, to give the required test solutions of 0.0015, 0.0050 and 0.010 mg/L. The control and the solvent control groups were maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 50 µl/L of dimethylformamide.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures.

Adult Daphnia were maintained in 150 mL glass vessels containing Elendt M7 medium 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 (Pseudokirchneriella subcapitata) and GM 300 fish 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:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Test temperature:
18-22 °C
pH:
6 to 9 +/- 1.5
Dissolved oxygen:
3 mg O2/L
Nominal and measured concentrations:
Nominal: 0 (blank control), 0 (solvent control), 0.0015, 0.0050 and 0.010 mg/L.
Measured: 0, 0, 0.00084, 0.0027 and 0.0062 mg/L
Details on test conditions:
The test vessels were preconditioned to the test solutions for 13 days prior to addition of the test organisms. The test preparations were renewed on Days 0, 1 and 8 of pre-conditioning. The concentration of the test item in the test preparations were verified by chemical analysis on Days 1, 3 and 4 during pre-conditioning.

For each concentration a single daphnid was placed in 100 mL of the test preparation in 180 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 and the solvent control groups were maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 50 µl/L of dimethylformamide.

The test preparations were renewed three 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 to 20 µL of an algal suspension (Pseudokirchneriella subcapitata) and approximately 20 µL of GM 300 fish food suspension on Days 0, 1, 2 and 3. 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.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Remarks:
immobilisation
Effect conc.:
>= 0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks:
parental animals
Remarks on result:
other: no effects at limit of water solubility
Key result
Duration:
21 d
Dose descriptor:
NOEC
Remarks:
reproduction
Effect conc.:
>= 0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}
Remarks on result:
other:
Remarks:
no effects at limit of water solubility
Key result
Duration:
21 d
Dose descriptor:
NOEC
Remarks:
growth
Effect conc.:
>= 0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
parental animals
Remarks on result:
other:
Remarks:
no effects at limit of water solubility
Duration:
21 d
Dose descriptor:
EC50
Remarks:
immobilisation
Effect conc.:
> 0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks:
parental animals
Remarks on result:
other:
Remarks:
no effects at limit of water solubility
Duration:
21 d
Dose descriptor:
EC50
Remarks:
reproduction
Effect conc.:
> 0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}
Remarks on result:
other:
Remarks:
no effects at limit of water solubility
Duration:
21 d
Dose descriptor:
EC50
Remarks:
growth
Effect conc.:
> 0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
parental animals
Remarks on result:
other:
Remarks:
no effects at limit of water solubility
Details on results:
Range-finding Test

Cumulative immobilization, sub lethal effects and the number of live young in the first brood data from the exposure of Daphnia magna to the test item during the range finding test were recorded. The data shows there was no reduction in the number of live young produced in the first brood at test concentrations of 0.0050 and 0.015 mg/L; however, a reduction was observed at the lowest test concentration of 0.0015 mg/L. Given that the reduced number of live young did not follow a dose response this reduction was considered not to be significant.

No immobilization was observed at any of the test concentrations. Sub lethal effects of exposure were observed in the solvent control and at test concentrations of 0.0050 and 0.015 mg/L. These responses were pale and small parental Daphnia. No sub lethal effects of exposure were observed at the test concentration of 0.0015 mg/L.

Based on this information test concentrations of 0.0015, 0.0050 and 0.010 mg/L were selected for the definitive test.

Chemical analysis of the fresh test preparations on Day 0 showed measured test concentrations to range from 0.00035 to 0.015 mg/L (between 24 and 98% of nominal concentrations). A decline in measured test concentration of the old test preparations on Day 2 was observed to between 0.00020 and 0.0070 mg/L (13% to 47% of nominal concentrations). Analysis of the 0.0050 mg/L test solution incubated without Daphnia and feed showed a measured concentration of 0.0035 mg/L compared to 0.0023 mg/L from the test solution containing the test organism indicating that some of the test item loss could be attributable to the presence of the biomass. Analysis of the fresh test preparation at 0.0015 mg/L showed a measured test concentration of 0.0015 mg/L (101% of the nominal concentration), therefore confirming that the error during preparation on Day 0 had been corrected.

Definitive test

Verification of test item concentrations
Based on the results of a preliminary range finding test, test concentrations of 0.0015, 0.0050 and 0.010 mg/L were selected for the definitive test.

Analysis of the fresh test preparations on Days 0, 7, 14 and 19 (see Annex 3) showed measured test concentrations to range from 0.00075 to 0.012 mg/L (50% to 117% of nominal concentrations). A decline in measured test concentration of the old test preparations on Days 2, 16 and 21 was observed to between 0.00048 and 0.0045 mg/L (26% to 45% of nominal concentrations) and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration in order to give a “worst case” analysis of the data.

Lethal Effects on the Parental Generation (P1)
No mortalities occurred in the control, solvent control and 0.00084 mg/L test groups throughout the test. A single parental mortality (immobilization) was recorded at the test concentrations of 0.0027 and 0.0062 mg/L; however, statistical analysis of the mortality data using the Fisher’s Exact test showed that the observed mortalities in the 0.0027 and 0.0062 mg/L test groups were not significantly different (P≥0.05) when compared to the control group. Accordingly these mortalities were determined to be inadvertent as their occurrence cannot be attributed to any potential test item effect. The ECx values were estimated by inspection of the data, whereas the NOEC and LOEC values (mortality) were determined using the Fisher Exact Test with Bonferroni Correction based on geometric mean measured test concentrations at 21 days.

Sub-lethal Effects on the Parental Generation (P1)
After 21 days the length of each surviving adult was determined.

The results showed that there were no statistically significant differences (P>=0.05) between the control and any of the test groups in terms of length of the daphnids after 21 days exposure to the test item.
The ECx values were estimated by inspection of the data, whereas the NOEC and LOEC values (body length) were determined using the Williams Multiple Sequential t-test Procedure based on geometric mean measured test concentrations at 21 days.

Effects on Reproduction
After 21 days there were no statistically significant differences between the control and the any of the test group in terms of the number of live young produced per adult. The ECx values were estimated by inspection of the data, whereas the NOEC and LOEC values (reproduction) were determined using the Williams Multiple Sequential t-test Procedure based on geometric mean measured test concentrations at 21 days.

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 media renewal showed the "filial" daphnids produced by all the test groups were in the same general condition as the young produced by the controls over the duration of the test. Young were first produced in the control test group on Day 7 of the test. There were no unhatched eggs or dead young observed in the control and treatment groups throughout the test.

Lowest Observed Effect Concentration
No significant effects for parental survival, number of live young produced or body length of the surviving adults were observed in any of the test concentrations over the duration of the test, hence the LOEC could not determined.

No Observed Effect Concentration
The NOEC was 0.0062 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 pooled control after 21 days. Given a theoretical water solubility of 0.0015 mg/l, the test item can be regarded as non-toxic within its water solubility limits.

Water Quality Criteria
Temperature was maintained at 19 °C to 21 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH. The water hardness of the test water used to prepare the test solution was observed to be in the range 240 to 256 mg/L as CaCO3 throughout the test. Throughout the test the light intensity was observed to be in the range 901 to 1099 lux. The water temperature was also recorded in the control vessel every hour using a Testo temperature logger.

Observations on Test Item Solubility
At the start and throughout the test all control and test solutions were observed to be clear
colorless solutions.

Conclusions
This study showed that there were no effects on parental immobilization, reproduction or body length at the highest attainable test concentration (including the limit of water solubility, 0.0015 mg/l) under experimental conditions.

Verification of Test Concentrations






















Nominal Test Concentration
(mg/L)



Geometric Mean Measured Test Concentration (mg/L)



0.0015



0.00084



0.0050



0.0027



0.010



0.0062



Validation Criteria









































 



Required



Actual



Control mortality



20%



0%



Mean number of live young per surviving adult (control group)



60 after 21 days



150



Coefficient of variation for control group*



25%



6%



No ephippia produced



0



0



Dissolved oxygen



>3 mg O2/L



≥3.7 mg O2/L



pH (control group)



6 to 9
Variation:
1.5



7.3 to 8.6
1.3






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


 




Lethal Effects on the Parental Generation (P1)








































Endpoint



Concentration


(mg/L)



Remarks



Mortality



EC10



>0.0062*



no effects up to water solubility limit



 



EC20



>0.0062*



 



EC50



>0.0062*



No Observed Effect Concentration (NOEC)



>=0.0062



Lowest Observed Effect Concentration (LOEC)



Not determined









*95% confidence limits not determined as no effects observed up to water solubility limit


 


 


Sub-lethal Effects on the Parental Generation (P1)



































Endpoint



Concentration


(mg/L)



Remarks



Body Length



EC10



>0.0062*



no effects up to water solubility limit



 



EC20



>0.0062*



 



EC50



>0.0062*



No Observed Effect Concentration (NOEC)



>=0.0062



Lowest Observed Effect Concentration (LOEC)



Not determined






 




*95% confidence limits not determined as no effects observed up to water solubility limit




 


Effects on Reproduction



































Endpoint



Concentration


(mg/L)



Remarks



Reproduction



EC10



>0.0062*



no effects up to water solubility limit



 



EC20



>0.0062*



 



EC50



>0.0062*



No Observed Effect Concentration (NOEC)



>=0.0062



Lowest Observed Effect Concentration (LOEC)



Not determined






*95% confidence limits not determined as no effects observed up to water solubility limit



Validity criteria fulfilled:
yes
Conclusions:
This study showed that there were no effects on parental immobilization, reproduction or body length at the highest attainable test concentration (including the limit of water solubility) under experimental conditions.
Executive summary:

The chronic toxicity of Santicizer 278 to daphnia was studied in a 21 -day test conducted in accordance with OECD 211. Initial test item preparation studies determined that the slow-stir method did not give appropriate or reproducible test item concentrations in the test solutions. Following trials, the use of a solvent (dimethylformamide) was found to be appropriate for producing test solutions that were reproducible and within a suitable range from the nominal concentrations. The water solubility of Santicizer 278 is 0.0015 mg/l and as such an extended limit test was performed at 0.0015, 0.050 and 0.01 mg/l (geometric mean measured concentrations of 0.00084, 0.0027 and 0.0062 mg/l respectively) to ensure that the test was performed at concentrations above the water solubility. This study showed that there were no effects on parental immobilization, reproduction or body length at the highest attainable test concentration (including the limit of water solubility, 0.0015 mg/l) under experimental conditions.

Description of key information

The chronic toxicity of Santicizer 278 to daphnia was studied in a 21 -day test conducted in accordance with OECD 211. Initial test item preparation studies determined that the slow-stir method did not give appropriate or reproducible test item concentrations in the test solutions. Following trials, the use of a solvent (dimethylformamide) was found to be appropriate for producing test solutions that were reproducible and within a suitable range from the nominal concentrations. The water solubility of Santicizer 278 is 0.0015 mg/l and as such an extended limit test was performed at 0.0015, 0.050 and 0.01 mg/l (0.00084, 0.0027 and 0.0062 mg/l respectively) to ensure that the test was performed at concentrations above the water solubility. This study showed that there were no effects on parental immobilization, reproduction or body length at the highest attainable test concentration (including the limit of water solubility, 0.0015 mg/l) under experimental conditions.

Key value for chemical safety assessment

Additional information