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Long-term toxicity to aquatic invertebrates

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Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
2018-01-12 to 2018-04-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Version / remarks:
Commission Regulation (EC) No 735/2017, Appendix 8, Official Journal of the European Union (EN) L 112 Volume 60, dated April 28, 2017
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
adopted October 02, 2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures"
Version / remarks:
December 15, 2000
Qualifier:
according to guideline
Guideline:
other: SANCO/3029/99 rev.4: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414
Version / remarks:
11/07/2000
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Storage Conditions at Test Facility:
At 20 ± 5 °C, in the dark
Analytical monitoring:
yes
Details on sampling:
One sample from the freshly prepared application solutions (in solvent DMF) and two sets of duplicate samples from the test media of all replicates of the only test concentration, the control, and the solvent control were taken prior to test start (equilibration phase).

One sample from the freshly prepared application solutions (in solvent DMF) and two sets of duplicate samples from the test media of all replicates of the only test concentration, the control, and the solvent control were taken at test start (day 0) and at three sampling points in the second, third and fourth calendar week (days 5, 7, 14 and 20).

One set of duplicate samples were diluted by a factor of two with acetonitrile, the second sample set remained undiluted.

Additional samples of the control and of the dilution solvent were taken at each sampling without any sample treatment.

Storage:
All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed. Afterwards the samples were again stored deep frozen (≤ -20 °C) and were kept stored up to the date of the final report.

Analyses:
The concentrations of the test item Matrilox LP101M were analysed in the diluted duplicate test media samples from all replicates of the only test concentration, and in one of the control and solvent control samples, from all sampling times (days 0, 7 (control and solvent control samples from day 5), 14 and 20). The second (undiluted) sample set was not analysed.
Vehicle:
yes
Remarks:
N,N-Dimethylformamide (DMF)
Details on test solutions:
Test Concentrations:
0.16 µg test item/L (in 100 µL DMF/L and 10 µL Tween 80/L), i.e. limit test.
Control:
In the control, test water was used without addition of the solvent, Tween 80 or the test item.
Solvent Control:
In the solvent control, test water was used with 100 µL DMF/L and 10 µL/L Tween 80.

Dosage of Test Item:
Concentrated stock solutions of 200 mg test item/L were prepared once a week by dissolving 10.2, 10.3, 10.7 and 10.3 mg test substance in 51, 51.5, 53.5 and 51.5 mL 100% DMF, respectively.
800 µL of these concentrated stock solutions were added up to a volume of 100 mL with 100% DMF to obtain application solutions with a concentration of 1.6 mg test item/L DMF. The application solutions were renewed once a week.
The dosage of the test item media, control and solvent control water were performed with tube pumps. The application solution (test item in DMF), the DMF (solvent control) and the test water were pumped in mixing vessels for ten minutes per hour. The pumps were pumping 48 mL/min ISO medium and 4.8 µL/min application solution (test item) or DMF (solvent control). Then the pumps paused for 50 minutes. In the mixing vessels (one per replicate) the application solution or solvent respectively and the test water were continuously mixed using a magnetic stirrer. For the control, test water was also pumped into mixing vessels and also stirred using a magnetic stirrer. The accuracy of the pumped volume was checked twice a week.

See in detail below the mixing for the treatment and control groups:
Treatment replicates: 4.8 µL/min (± 10%) of the application solution were mixed with 48 mL/min (± 10%) of test water (ISO medium) to prepare the test medium of the test concentration of 0.16 µg test item/L.
Solvent control replicates: For the solvent control 4.8 µL/min (± 10%) 100% DMF were mixed with 48 mL/min (± 10%) of test water (ISO medium).
Control replicates: For the control 48 mL/min (± 10%) of test water (ISO medium) were used.
Tween 80 was dosed directly into the test water for the treatment and the solvent control with a concentration of 10 µL Tween 80/L test water. No Tween 80 was added to the control. After six days, the addition of Tween 80 to the test water was discontinued for the solvent control and the treatment group. However, from Day 7 onwards Tween 80 addition was resumed after consulting the study monitor because several animals of solvent control (replicates 2, 3, and 4) and treatment (replicate 1) were trapped at the surface. Tween 80 at concentration of 10 µL/L was added until the end of the study to the solvent control and the test concentration.
The accuracy of the dosage was checked 4 days before start of the test. The prepared test media flowed in the test aquaria via tubes. The excess of test media left the aquaria via an overflow pipe.

Appearance of the Test Item in the Test Media: No remarkable observations throughout the study.
Test organisms (species):
Daphnia magna
Details on test organisms:
Species: Daphnia magna (Straus), clone 5
Age at Test Start: 1.5 to 23 hours old
Sex: Female
Origin: The Daphnia introduced in the test were taken from ibacon's in-house laboratory culture.
Breeding Conditions: The Daphnia were bred in the laboratories of ibacon under similar temperature and light conditions as in the test. The cultivation of the parental Daphnia was performed in ISO medium (see IUCLID section "Any other information on materials and methods inc. tables" for details). The test organisms were not first brood progeny. The Daphnia in the stock culture were fed at least all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon.
Acclimatisation: Was not necessary, since the test was performed in the same medium as the culturing.
Test type:
flow-through
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Post exposure observation period:
no
Hardness:
160 to 214 mmol/L
Test temperature:
19.3 to 20.4 °C
pH:
7.1 to 8.9; the pH varied by more than 1.5 units, but this does not invalidate the test, because all validity criteria were met.
Accoding to the study plan, pH sould be between 6 - 9 during the whole test and not vary by more than 1.5 units. The range was met, the higher variation during the study may have been caused by Algae given as a food source to the Daphnia, which may cause an increase of the pH.
Dissolved oxygen:
7.7 to 11.2 mg/L
Salinity:
not applicable
Conductivity:
not specified
Nominal and measured concentrations:
Limit test with one treatment group:
0.16 µg test item/L (in 100 µL DMF/L and 10 µL Tween 80/L), corresponding to the following mean measured concentrations of the test item per replicate, corrected for recovery of fortified samples:
0.150, 0.176, 0.179, and 0.117 μg test item/L, resulting in an overall mean of 0.156 µg/L for the treatment group.
This corresponds to 97% of the nominal limit concentration. Accordingly, all (no)effect concentrations are given based on the nominal limit concentration of 0.160 µg/L according to OECD 211, paragraph 50.
Justification:
Based on results from the preliminary test and the determined water solubility of 0.078 µg/L in ISO medium (Sonntag, 2017) no toxicity of the test item up to the saturation concentration in water was expected. Therefore, it was decided that a limit test as foreseen according to OECD 211, paragraph 36 would be most appropriate in this case. Accordingly, the actually applied nominal test item concentrations of 0.16 µg test item /L (equalling to the rounded value of water solubility times a safety factor of 2 as a reasonable compromise between trying to avoid a relevant non-dissolved fraction on the one hand and minimizing the risk of failing a saturated solution in case of analytical recoveries somewhat below the nominal concentration) was used as the limit concentration. While reducing the normally required 5 test item concentrations to one, four replicates with 10 animals, each, were tested for the only treatment an the controls. Further, analytical monitoring was extended beyond the minimum required according to OECD 211 (one replicate per concentration level), in that regularly all four replicates per treatment / controls were analysed. For details on considerations regarding test design see IUCLID section "Any other information on materials and methods incl. tables".
Details on test conditions:
Please see IUCLID section "Any other information on materials and methods incl. tables" for details.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.16 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Remarks:
analytically determined test item concentration within +/- 20% of nominal
Basis for effect:
reproduction
Remarks on result:
other: corresponding to the saturation concentration in test medium
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 0.16 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Remarks:
analytically determined test item concentration within +/- 20% of nominal
Basis for effect:
reproduction
Remarks on result:
other: corresponding to the saturation concentration in test medium
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.16 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Remarks:
analytically determined test item concentration within +/- 20% of nominal
Basis for effect:
mortality
Remarks on result:
other: corresponding to the saturation concentration in test medium
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 0.16 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Remarks:
analytically determined test item concentration within +/- 20% of nominal
Basis for effect:
mortality
Remarks on result:
other: corresponding to the saturation concentration in test medium
Details on results:
Please see IUCLID sections "Any other information on results incl. tables" and "Overall remarks, attachments" for details.
Results with reference substance (positive control):
no reference substance
Reported statistics and error estimates:
Please see IUCLID section "Any other information on materials and methods incl. tables".

Validity Criteria of the Study

Survival Rate of Adult Daphnia in the Control:       

control: 100 %; solvent Control: 97.5 %; pooled control 98.8 %; validity criterion was met.

Mean Number of Live Offspring in the Control after 21 Days:       

offspring per surviving adult Daphnia:

control: 131.1; solvent control: 143.0 and pooled control: 136.9; validity criterion was met.

      

Biological Results       

Reproduction per Survived Parent (Cumulative Offspring per Survived Parent):

21-day NOEC       ≥ 0.16 µg test item/L

21-day LOEC       > 0.16 µg test item/L

21-day EC50 (95 % CI)       > 0.16 µg test item/L (n.d.)

21-day EC20 (95 % CI)       > 0.16 µg test item/L (n.d.)

21-day EC10 (95 % CI)       > 0.16 µg test item/L (n.d.)

      

Mortality:       

21-day NOEC       ≥ 0.16 µg test item/L

21-day LOEC       > 0.16 µg test item/L

21-day EC50 (95 % CI)       > 0.16 µg test item/L (n.d.)

CI: Confidence interval, n.d.: not determinable; values refer to nominal concentrations

      

Mortality of Adults:       

No significant difference between the control and the solvent control was detected (fisher´s exact test) and therefore both controls were pooled for the statistical analysis. In the control all Daphnia survived until the end of the test, in the solvent control and the only test item concentration of 0.16 µg test item/L one Daphnia died during the test. Thus, the mortality of Daphnia magna after 21 days at the only concentration of 0.16 µg test item/L was not significantly different from the pooled control (see Table B-1, IUCLID section "Overall remarks, attachments").

Reproduction:

There was no significant difference between the control and the solvent control. Therefore the two controls were pooled for statistical analysis.

Fisher´s Exact Binominal Test was used to test for significant differences in mortality of parental daphnia between the pooled control and the treatment. No significant difference was observed; therefore, observed mortality is concluded to be unrelated to the test item but "inadvertant" according to OECD 211, paragraph 51. Accordingly, the relevant endpoint is reproductive output per surviving parent and all statistical analyses are based on the number of offspring by all surviving parents.

The total numbers of live offspring produced by each introduced adult for each treatment group is given in Table B-2, IUCLID section "Overall remarks, attachments".

The total numbers of live offspring produced by each surviving adult for each treatment group as used for the statistical analysis is given in Table B-3, IUCLID section "Overall remarks, attachments".

The reproduction of the Daphnia was 131 ± 12.1 (mean ± SD) live offspring per surviving adult in the control, 143.0 ± 21.3 in the solvent control and 136.9 ± 16.7 in the pooled control.

The coefficient of variation around the mean number of living offspring produced per surviving parent Daphnia in the control was 9.3%, in the solvent control 14.9 and in the pooled control 12.1%. According to the results of a Two sample t-test (one-sided smaller, alpha = 0.05) no significant toxic effect of the test item on the mean reproduction was determined for the only test concentration of 0.16 µg test item/L. The EC50 of the reproduction was determined to be > 0.16 µg test item/L.

Date of First Brood:       

The first offspring released from their parent animals were recorded in the control, the solvent control and the test concentration of 0.16 µg test item/L at the observation Day 10 (see Table B-4, IUCLID section "Overall remarks, attachments").

Signs of Intoxication:       

No signs of intoxication were observed in the test animals during the test.

      

Analytical Results

Table 1: Summary of Analytical Results

Sample Description
[µg test item/L]

Mean measured concentrations
[µg test item/L]

Mean measured concentrations*
[µg test item/L]

% of nominal*1

n

Control

<LOD

<LOD

n.a.

16

Solvent control

<LOD

<LOD

n.a.

16

0.16

0.113

0.150

94

8

0.16

0.132

0.176

110

8

0.16

0.135

0.179

112

8

0.16

0.088

0.117

73

8

(1) mean value of all measured samples per treatment group;

RSD: relative standard deviation per treatment group;

n: number of analysed samples;

n.a.: not applicable;

italic: For values <LOQ, half the LOQ was used for calculating measured concentrations according to OECD Guidance Document No. 23;

*) Results corrected for recoveries of fortified samples (recovery 75 %, correction factor 1.33)

Mean Recovery in the Test Samples:       

73 % (n = 32)

97 % (n = 32)*

For details see Table A-1, IUCLID section "Overall remarks, attachments".

*) Values corrected for recoveries obtained from fortified samples (recovery 75 %, correction factor 1.33). Details on results for fortified samples are given in Table A-2, IUCLID section "Overall remarks, attachments".

Validity Criteria of the Analytical Part (based on SANCO/3029 (SANCO/3029/99 rev.4 11/07/00)

Specificity:       

No or no significant (< 30 % for re-analysis) interference of total peak area for the target analyte was found (target value: ≤30%).

Linearity:       

Calibration Range:

0.025 – 0.175 µg test item/L;

Linearity of Response:

Correlation of peak area of different standard solutions with their corresponding concentrations, using a linear regression - Correlation Coefficient: r = 0.9967 at least (target value: >=0.99)

Calibration Curves:

y = 408366 * x – 741

Re-analysis:

y = 639162 * x + 14362

Accuracy and Precision:       

Mean Recovery Rates in the Fortified Samples:

75 % (n = 10, RSD 5 %) - target values: 70%-110% for accuracy; RSD ≤20% for precision.

The values found for the precision (RSD) and for the accuracy (mean recovery rate) are acceptable (for details see Table A-2).

Conclusion:       The validity criteria for the analytical method have been met.

Overall conclusions       

The effects of the test item Matrilox LP101M on the mortality and reproduction of Daphnia magna was determined during an exposure period of 21 days. At the saturation level in test medium (corresponding to the nominal limit concentration of 0.16 µg test item /L) not any toxic effect of the test item relative to controls could be observed:

The 21 day NOEC was determined to be ≥ 0.16 µg test item/L for the reproduction and ≥ 0.16 µg test item/L for the mortality of the adult test animals.

The EC50 for the reproduction was > 0.16 µg test item/L.

The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to nominal values since the concentrations of the test item were within ± 20 % of the nominal concentrations during the test. The measured values were corrected for the recoveries of the fortified samples (recovery 75 %, correction factor 1.33).

Validity criteria fulfilled:
yes
Conclusions:
Up to the saturation level in test medium (corresponding to the nominal limit concentration of 0.16 µg test item /L, confirmed by analytical monitoring) not any toxic effect of the test item on Daphnia magna reproduction or parental animals could be observed relative to controls (OECD 211; GLP; 2018):
NOEC (Daphnia magna; 21 days; reproduction) ≥ saturation level in water;
NOEC (Daphnia magna; 21 days; adult mortality) ≥ saturation level in water;
LOEC / EC50 (Daphnia magna; 21 days; reproduction and adult mortality) > saturation level in water.
Executive summary:

The purpose of this study was to evaluate the influence of the test item Matrilox LP101M (Trimethylolpropane trinonanoate) on the mortality and reproduction of Daphnia magna during an exposure period of 21 days. Young Daphnia (< 24 hours old) were exposed in a flow through test to aqueous test media containing the test item at the only concentration of nominal 0.16 µg test item/L (limit test). The test concentration was chosen as the water solubility multiplied with a safety factor of 2 based on analytical recoveries observed in the preliminary test. Because no effects were determined in this preliminary test up to and above the water solubility, a limit test was performed to demonstrate that the test item causes no effects to the test organism at the saturation concentration in water. The mortality, number of offspring, and symptoms of intoxication were compared with corresponding parameters in the control.

The test method of application and the test species Daphnia magna are recommended by the test guidelines.

The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.

The test was performed according to the following guidelines, compliant with GLP:

- EU Method C.20.: "Daphnia magna Reproduction Test", Commission Regulation (EC) No 735/2017, Appendix 8, Official Journal of the European Union (EN) L 112 Volume 60, dated April 28, 2017

- OECD Guideline for Testing of Chemicals, No. 211: "Daphnia magna Reproduction Test", adopted October 02, 2012

- OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures", December 15, 2000

- SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414 for assessment of validity of analytical data.

This study encompassed 1 test item concentration of 0.16 µg test item/L (in 100 µL Dimethylformamide (DMF)/L and 10 µL Tween 80/L), a solvent control (100 µL DMF/L and 10 µL Tween 80/L) and a control, with 4 replicates, each containing 10 Daphnia in separate cages. Tween 80 below the critical micelle concentration (CMC) had to be added to prevent daphnids from being trapped on the surface in the solvent control as well as the treatment. This phenomenon was not observed in the control without solvent and thus seems to be related to the presence of DMF (100 µL/L) in these groups. Endpoints assessed were mortality of adults and number of offspring per survived parent (no symptoms of intoxication observed).

The quantification of the test item Matrilox LP101M in the test samples was performed using liquid chromatography (HPLC) with MS/MS detection. All four replicates were regularly assessed at day 0, 7, 14 and 20. Further, sampling at day 4 before introduction of animals confirmed successful equilibration of the final test item concentration for all four replicates of the limit concentration.

In the test media, 97 % of the nominal test concentration was found (mean values of nominal 0.16 µg/L, corrected for the recoveries of 75 % in the fortified samples, correction factor 1.33) throughout the test (arithmetic mean value). Correct dosing of the test item could be demonstrated. Accordingly, all reported results refer to nominal values since the concentrations of the test item were within ± 20 % of the nominal concentrations during the test.

Biological results:

At the saturation level in test medium (corresponding to the nominal limit concentration of 0.16 µg test item /L) not any toxic effect of the test item relative to controls could be observed:

The 21 day NOEC was determined to be ≥ 0.16 µg test item/L for the reproduction and ≥ 0.16 µg test item/L for the mortality of the adult test animals.

The LOEC / EC50 for the reproduction was > 0.16 µg test item/L.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2017 to June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Version / remarks:
Commission Regulation (EC) No 735/2017, Appendix 8, Official Journal of the European Union (EN) L 112 Volume 60, dated April 28, 2017
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
adopted October 02, 2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures"
Version / remarks:
December 15, 2000
Qualifier:
according to guideline
Guideline:
other: SANCO/3029/99 rev.4: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414
Version / remarks:
11/07/2000
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Storage Conditions at Test Facility:
At 20 ± 5 °C, in the dark
Analytical monitoring:
yes
Details on sampling:
One sample from the freshly prepared application solutions (in solvent DMF) and two sets of duplicate samples from the test media of all replicates of the prepared test concentrations, the control, and the solvent control were taken prior to test start (equilibration phase).

One sample from the freshly prepared application solutions (in solvent DMF) and two sets of duplicate samples from the test media of all replicates of the only test concentration, the control, and the solvent control were taken at test start (day 0) and at three sampling points in the second, third and fourth calendar week (days 5, 7, 14 and 20).

One set of duplicate samples were diluted by a factor of two with acetonitrile, the second sample set remained undiluted.

Additional samples of the control and of the dilution solvent were taken at each sampling without any sample treatment.

Storage:
All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed. Afterwards the samples were again stored deep frozen (≤ -20 °C) and were kept stored up to the date of the final report.

Analyses:
The concentrations of the test item Matrilox LP101M were analysed in the diluted duplicate test media samples from all replicates of the only test concentration, and in one of the control and solvent control samples, from all sampling times (days 0, 7 (control and solvent control samples from day 5), 14 and 20). The second (undiluted) sample set was not analysed.
Vehicle:
yes
Remarks:
N,N-Dimethylformamide (DMF)
Details on test solutions:
Test Concentrations:
270, 135, 67.5, 33.8 and 16.9 μg test item/L (each in 100 μL DMF/L) and 10 µL Tween 80/L, a solvent control (100 μL DMF/L) and and 10 µL Tween 80/L, and a control; corresponding to the following mean measured concentrations of 411, 183, 59.0, 33.5 and 20.9 μg test item/L (each in 100 μL DMF/L). The nominal concentrations were chosen according to the water solubility limit.
Control:
In the control, test water was used without addition of the solvent or the test item.
Solvent Control:
In the solvent control, test water was used with 100 μL DMF/L.

Dosage of Test Item:
Concentrated stock solutions of 2.7 g test item/L were prepared by dissolving 108.0, 109.1, 113.8 mg test item into 40.0, 40.4, 42.15 mL 100 % DMF, respectively by stirring for 5 to 10 minutes. The lower concentrated stock solutions of 1.35, 0.675, 0.338 and 0.169 g test item/L were prepared by diluting the stock solution of 2.7 g test item/L with adequate volumes of 100 % DMF. The stock solutions were renewed once a week.
The dosage of the test item media, control and solvent control water were performed with tube pumps. The application solution (concentrations), the DMF (solvent control) and the test water were pumped in mixing vessels for ten minutes per hour. The pumps were pumping 48 mL/min ISO medium and 4.8 µL/min stock solution (test item) or DMF (solvent control). Then the pumps paused for 50 minutes. In the mixing vessels the application solution or solvent respectively and the test water were continuously mixed using a magnetic stirrer. For the control, test water was also pumped into mixing vessels and also stirred using a magnetic stirrer. The accuracy of the pumped volume was checked twice a week.
See in detail below the mixing for the different treatments:
4.8 µL/min (± 10%) of the respective stock solution was mixed with 48 mL/min (± 10%) of test water (ISO medium) to prepare the test medium of the test concentration respective test item concentration. For the solvent control 4.8 µL/min (± 10%) 100% DMF were mixed with 48 mL/min (± 10%) of test water (ISO medium). For the control 48 mL/min (± 10%) of test water (ISO medium) were used.
Tween 80 was dosed directly into the test water for the concentrations and the solvent control with a concentration of 10 µL Tween 80/L test water. No Tween 80 was added to the control. Tween 80 at a concentration of 10 µL/L was added until the end of the study to the solvent control and the test concentrations. Tween 80 was used to reduce the surface tension occurring in the flow through tests using DMF.

Appearance of the Test Item in the Test Media: No remarkable observations throughout the study.
Test organisms (species):
Daphnia magna
Details on test organisms:
Species: Daphnia magna (Straus), clone 5
Age at Test Start: 1.5 to 23 hours old
Sex: Female
Origin: The Daphnia introduced in the test were taken from ibacon's in-house laboratory culture.
Breeding Conditions: The Daphnia were bred in the laboratories of ibacon under similar temperature and light conditions as in the test. The cultivation of the parental Daphnia was performed in ISO medium (see IUCLID section "Any other information on materials and methods inc. tables" for details). The test organisms were not first brood progeny. The Daphnia in the stock culture were fed at least all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon.
Acclimatisation: Was not necessary, since the test was performed in the same medium as the culturing.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
no
Hardness:
196 to 249 CaCO3 mg/L
Test temperature:
19.7 to 21.3°C
pH:
7.7 to 8.3; and thus the pH-value did not vary by more than 1.5 units
Dissolved oxygen:
8.2 to 9.3 mg/L
Salinity:
In deionised water analytical grade salts and additives were added at the following nominal concentrations (ISO MEDIUM):
CaCl2 × 2H2O 2.0 mmol/L (= 294 mg/L)
MgSO4 × 7H2O 0.5 mmol/L (= 123 mg/L)
NaHCO3 0.75 mmol/L (= 65 mg/L)
KCl 0.075 mmol/L (= 5.8 mg/L)
Additionally added will be the Trace Element:
Na2SeO3 2.2 μg/L
And Vitamins:
Thiamin HCl 75.0 μg/L
Cyanocobalamin (B12) 1.0 μg/L
Biotin (B6) 0.75 μg/L
Conductivity:
not specified
Nominal and measured concentrations:
270, 135, 67.5, 33.8 and 16.9 µg test item/L (each in 100 µL DMF/L), a solvent control (100 µL DMF/L) and a control; corresponding to the following mean measured concentrations of 411, 183, 59.0, 33.5 and 20.9 µg test item/L (each in 100 µL DMF/L).
Details on test conditions:
Please see IUCLID section "Any other information on materials and methods incl. tables" for details.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 411 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 411 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 411 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 411 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 411 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
reproduction
Remarks on result:
other:
Remarks:
Confidence interval 95%
Details on results:
Please see IUCLID sections "Any other information on results incl. tables" and "Overall remarks, attachments" for details.
Results with reference substance (positive control):
no reference substance
Reported statistics and error estimates:
Please see IUCLID section "Any other information on materials and methods incl. tables".

Validity Criteria of the Study

Survival Rate of Adult Daphnia in the Control and Solvent Control:

100 % and 100 %, respectively; validity criterion was met

Mean Number of Live Offspring in the Control and Solvent Control after 21 Days:

125.6 and 115.8 per surviving adult Daphnia, respectively; validity

      

Biological Results       

Reproduction per Survived Parent (Cumulative Offspring per Survived Parent): :

21-day NOEC ≥ 411 μg test item/L

21-day LOEC > 411 μg test item/L

21-day EC50 (95 % CI) > 411 μg test item/L (n.d.)

21-day EC20 (95 % CI) > 411 μg test item/L (n.d.)

21-day EC10 (95 % CI) > 411 μg test item/L (n.d.)

Mortality:

21-day NOEC ≥ 411 μg test item/L

21-day LOEC > 411 μg test item/L

21-day EC50 (95 % CI) > 411 μg test item/L (n.d.)

CI: Confidence interval, n.d.: not determinable; values refer to mean measured concentrations

      

Mortality of Adults:       

The Fisher´s Exact Binominal Test with Bonferroni Correction revealed no significant trend in mortality. At the control, the solvent control and the test item concentration of 20.9, 183 and 411 μg test item/L, all Daphnia survived until the end of the test. In the test concentrations of 33.5 and 59.0 μg test item/L one animal died during the test. Thus, the mortality of Daphnia magna after 21 days was not significantly affected by the test item and was therefore determined to be higher than the highest tested concentration of 411 μg test item/L.

Reproduction:

Since the Cochran-Armitage trend test revealed no significant trend in mortality all statistical analyses are based on the number of living offspring by all survived parents.

There was no significant difference between the control and the solvent control. According to OECD 54 the concentrations were tested against the solvent control.

The coefficient of variation around the mean number of living offspring produced per surviving parent Daphnia in the control was 19.8 % and the solvent control 25.4 %. According to the results of a Dunnett´s t-test (one-sided smaller, α = 0.05) no significant toxic effect of the test item on the mean reproduction compared to the solvent control was determined at the test concentration of 20.9, 183 and 411 μg test item/L. At the concentrations 33.5 and 59.0 μg test item/L, the mean reproduction of surviving Daphnia was statistically significantly reduced compared to the control. The EC50 of the reproduction was determined from the raw data to be > 411 μg test item/L.

The two concentrations 33.5 and 59.0 μg test item/L showed a very low number of offspring for all single kept Daphnia. It was observed during the study that the Daphnia at concentration 33.5 and 59.0 μg test item/L were much smaller than the Daphnia from both controls and the test concentrations. An explanation might be in a wrong constitution of the test water.

The test water for the concentrations and the control came from tons capturing a volume each of 90 Litre. The test water was prepared three times per week with ISO water from a tank, where ISO water is automatized prepared. Additionally added by hand are the trace elements (see test water 6.5). The concentration 33.5 and 59.0 μg/L received test water from the same ton. So it can be assumed that early in the developing phase the test water was prepared not correctly and therefore the daphnia of the concentrations 33.5 and 59.0 μg test tem/L were smaller and show a much lower reproduction than the higher concentrations 183 and 411 μg test item/L.

Date of First Brood:       

The first offspring released from their parent animals were recorded in the control, the solvent control and the test concentrations 20.9 and 183 μg test item/L at the observation on Day 10. At the test concentrations of 59.0 and 411 μg test item/L the first offspring was observed on Day 11 and at the concentration of 335 μg test item/L at Day 14.

Signs of Intoxication:       

With exception of the reported mortality and the reduced reproduction, no other signs of intoxication were observed in the test animals during the test. At the concentrations 33.5 and 59.0 μg test item/L the Daphnia were smaller compared to the controls and the other test concentrations.

      

Analytical Results

Table 1: Summary of Analytical Results

Sample Description
[µg test item/L]

Mean measured concentrations
[µg test item/L]

Mean measured concentrations*
[µg test item/L]

% of nominal*1

n

Control

<LOD

<LOD

n.a.

8

Solvent control

<LOD

<LOD

n.a.

8

16.9

124

23

20.9

8

33.8

99

29

33.5

8

67.5

87

12

59.0

8

135

136

40

183.0

8

 270

 152  43  411.0  8

(1) mean value of all measured samples per treatment group;

RSD: relative standard deviation per treatment group;

n: number of analysed samples;

n.a.: not applicable;

Mean Recovery in the Test Samples:       

120 % (n = 40, RSD 39%)

97 % (n = 32)*

For details see Table A-1, IUCLID section "Overall remarks, attachments".

*) where the tabulated results represent rounded results calculated on the exact raw data.

Details on results for fortified samples are given in Table A-2, IUCLID section "Overall remarks, attachments".

Validity Criteria of the Analytical Part (based on SANCO/3029 (SANCO/3029/99 rev.4 11/07/00)

Specificity:       

No interference of total peak area for the target analyte was found.

Linearity:       

Calibration Range:

Two calibration curves were used in order to cover the wide concentration range of 2 – 125 μg test item/L with high accuracy.

1. 2 – 75 μg test item/L

2. 2 – 125 μg test item/L

Linearity of Response:

Correlation of peak area of different standard solutions with their corresponding concentrations, using a linear regression - Correlation Coefficient: r = 0.9980 at least (target value: >=0.99)

Calibration Curves:

y = 451 * x – 113

Re-analysis:

y = 727 * x -86

Accuracy and Precision:       

Mean Recovery Rates in the Fortified Samples:

82 % (n = 15, RSD 10 %) - target values: 70%-110% for accuracy; RSD ≤20% for precision.

The values found for the precision (RSD) and for the accuracy (mean recovery rate) are acceptable (for details see Table A-2).

Conclusion:       The validity criteria for the analytical method have been met.

Overall conclusions       

The effects of MATRILOX LL101M on the mortality and reproduction of Daphnia magna was determined during an exposure period of 21 days. The 21-day NOEC was determined to be ≥ 411 μg test item/L for the reproduction and ≥ 411 μg test item/L for the mortality of the adult test animals. The EC50 for the reproduction was > 411 μg test item/L

The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to measured values since the concentrations of the test item were not within ± 20% of the nominal concentrations during the test

Validity criteria fulfilled:
yes
Conclusions:
The effects of MATRILOX LL101M on the mortality and reproduction of Daphnia magna was determined during an exposure period of 21 days. The 21-day NOEC was determined to be ≥ 411 μg test item/L for the reproduction and ≥ 411 μg test item/L for the mortality of the adult test animals. The EC50 for the reproduction was > 411 μg test item/L
The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to measured values since the concentrations of the test item were not within ± 20% of the nominal concentrations during the test.
Executive summary:

The purpose of this study was to evaluate the influence of the test item Matrilox LL101M (Trimethylolpropane Carboxylic Acids, C5 -9, triester) on the mortality and reproduction of Daphnia magna during an exposure period of 21 days. Young Daphnia (< 24 hours old) were exposed in a flow through test to aqueous test media containing the test item at the concentrations 270, 135, 67.5, 33.8 and 16.9 µg of test item/L. The nominal concentrations were chosen according to the water solubility limit. The mortality, number of offspring, and symptoms of intoxication were compared with corresponding parameters in the control.

The test method of application and the test species Daphnia magna are recommended by the test guidelines.

The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.

The test was performed according to the following guidelines, compliant with GLP:

- EU Method C.20.: "Daphnia magna Reproduction Test", Commission Regulation (EC) No 735/2017, Appendix 8, Official Journal of the European Union (EN) L 112 Volume 60, dated April 28, 2017

- OECD Guideline for Testing of Chemicals, No. 211: "Daphnia magna Reproduction Test", adopted October 02, 2012

- OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures", December 15, 2000

- SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414 for assessment of validity of analytical data.

This study encompassed 5 test item concentrations of test item/L (in 100 µL Dimethylformamide (DMF)/L and 10 µL Tween 80/L), a solvent control (100 µL DMF/L and 10 µL Tween 80/L) and a control, This study encompassed 5 test concentrations, a solvent control and a control with 10 replicates, each containing one Daphnia in separate cages. Tween 80 below the critical micelle concentration (CMC) had to be added to prevent daphnids from being trapped on the surface in the solvent control as well as the treatment. This phenomenon was not observed in the control without solvent and thus seems to be related to the presence of DMF (100 µL/L) in these groups. Endpoints assessed were mortality of adults and number of offspring per survived parent (no symptoms of intoxication observed).

The quantification of the test item Matrilox LL101M in the test samples was performed using liquid chromatography (HPLC) with MS/MS detection. All replicates were regularly assessed at day 0, 7, 14 and 20. Further, sampling at day 4 before introduction of animals confirmed successful equilibration of the final test item concentration for all four replicates of the limit concentration.

In the test media the recoveries ranged from 73 to 267 % of the nominal test concentrations (all test concentrations considered). The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to measured values since the concentrations of the test item were not within ± 20% of the nominal concentrations during the test.

Biological results:

With exception of the reported mortality and the reduced reproduction, no other signs of intoxication were observed in the test animals during the test. At the concentrations 33.5 and 59.0 μg test item/L the Daphnia were smaller compared to the controls and the other test concentrations. An explanation might be in a wrong constitution of the test water. The Fisher´s Exact Binominal Test with Bonferroni Correction revealed no significant trend in mortality. At the control, the solvent control and the test item concentration of 20.9, 183 and 411 μg test item/L, all Daphnia survived until the end of the test. In the test concentrations of 33.5 and 59.0 μg test item/L one animal died during the test. Thus, the mortality of Daphnia magna after 21 days was not significantly affected by the test item and was therefore determined to be higher than the highest tested concentration of 411 μg test item/L

The 21-day NOEC was determined to be ≥ 411 μg test item/L for the reproduction and ≥ 411 μg test item/L for the mortality of the adult test animals. The EC50 for the reproduction was > 411 μg test item/L

Description of key information

Two experimental studies according to OECD 211 are available, one on the substance and a second one on the analogue Trimethylolpropane Tripelargonate (see RA justification in the section 13).

According to the available key study on the target substance, the effects of MATRILOX LL101M on the mortality and reproduction of Daphnia magna was determined during an exposure period of 21 days. The 21-day NOEC was determined to be ≥ 411 μg test item/L for the reproduction and ≥ 411 μg test item/L for the mortality of the adult test animals. The EC50 for the reproduction was > 411 μg test item/L

The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part.

In the case of the supporting study on the analogue Trimethylolpropane Tripelargonate, up to the saturation level in test medium (corresponding to the nominal limit concentration of 0.16 µg test item /L, confirmed by analytical monitoring) not any toxic effect of the test item on Daphnia magna reproduction or parental animals could be observed relative to controls (OECD 211; GLP; 2018):

NOEC (Daphnia magna; 21 days; reproduction) ≥ saturation level in water;

NOEC (Daphnia magna; 21 days; adult mortality) ≥ saturation level in water;

LOEC / EC50 (Daphnia magna; 21 days; reproduction and adult mortality) > saturation level in water.

Because NOEC / LOEC values are solely determined by the very low water solubility of the test item (no toxicity observed up to the water solubility limit) and are thus "greater than" values, no key values for chemical safety assessment are given below, because no hazard was identified.

Key value for chemical safety assessment

Additional information

For long-term toxicity to aquatic invertebrates, two reliable studies are available.

The purpose of the key study was to evaluate the influence of the test item Matrilox LL101M (Trimethylolpropane Carboxylic Acids, C5 -9, triester) on the mortality and reproduction of Daphnia magna during an exposure period of 21 days. Young Daphnia (< 24 hours old) were exposed in a flow through test to aqueous test media containing the test item at the concentrations 270, 135, 67.5, 33.8 and 16.9 µg of test item/L. The nominal concentrations were chosen according to the water solubility limit. The mortality, number of offspring, and symptoms of intoxication were compared with corresponding parameters in the control.

The test method of application and the test species Daphnia magna are recommended by the test guidelines.

The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.

The test was performed according to the following guidelines, compliant with GLP:

- EU Method C.20.: "Daphnia magna Reproduction Test", Commission Regulation (EC) No 735/2017, Appendix 8, Official Journal of the European Union (EN) L 112 Volume 60, dated April 28, 2017

- OECD Guideline for Testing of Chemicals, No. 211: "Daphnia magna Reproduction Test", adopted October 02, 2012

- OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures", December 15, 2000

- SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414 for assessment of validity of analytical data.

This study encompassed 5 test item concentrations of test item/L (in 100 µL Dimethylformamide (DMF)/L and 10 µL Tween 80/L), a solvent control (100 µL DMF/L and 10 µL Tween 80/L) and a control, This study encompassed 5 test concentrations, a solvent control and a control with 10 replicates, each containing one Daphnia in separate cages. Tween 80 below the critical micelle concentration (CMC) had to be added to prevent daphnids from being trapped on the surface in the solvent control as well as the treatment. This phenomenon was not observed in the control without solvent and thus seems to be related to the presence of DMF (100 µL/L) in these groups. Endpoints assessed were mortality of adults and number of offspring per survived parent (no symptoms of intoxication observed).

The quantification of the test item Matrilox LL101M in the test samples was performed using liquid chromatography (HPLC) with MS/MS detection. All replicates were regularly assessed at day 0, 7, 14 and 20. Further, sampling at day 4 before introduction of animals confirmed successful equilibration of the final test item concentration for all four replicates of the limit concentration.

In the test media the recoveries ranged from 73 to 267 % of the nominal test concentrations (all test concentrations considered). The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to measured values since the concentrations of the test item were not within ± 20% of the nominal concentrations during the test.

Biological results:

With exception of the reported mortality and the reduced reproduction, no other signs of intoxication were observed in the test animals during the test. At the concentrations 33.5 and 59.0 μg test item/L the Daphnia were smaller compared to the controls and the other test concentrations. An explanation might be in a wrong constitution of the test water. The Fisher´s Exact Binominal Test with Bonferroni Correction revealed no significant trend in mortality. At the control, the solvent control and the test item concentration of 20.9, 183 and 411 μg test item/L, all Daphnia survived until the end of the test. In the test concentrations of 33.5 and 59.0 μg test item/L one animal died during the test. Thus, the mortality of Daphnia magna after 21 days was not significantly affected by the test item and was therefore determined to be higher than the highest tested concentration of 411 μg test item/L

The 21-day NOEC was determined to be ≥ 411 μg test item/L for the reproduction and ≥ 411 μg test item/L for the mortality of the adult test animals. The EC50 for the reproduction was > 411 μg test item/L

The purpose of the supporting study was to evaluate the influence of the test item Matrilox LP101M (Trimethylolpropane trinonanoate) on the mortality and reproduction of Daphnia magna during an exposure period of 21 days. Young Daphnia (< 24 hours old) were exposed in a flow through test to aqueous test media containing the test item at the only concentration of nominal 0.16 µg test item/L (limit test). The test concentration was chosen as the water solubility multiplied with a safety factor of 2 based on analytical recoveries observed in the preliminary test. Because no effects were determined in this preliminary test up to and above the water solubility, a limit test was performed to demonstrate that the test item causes no effects to the test organism at the saturation concentration in water. The mortality, number of offspring, and symptoms of intoxication were compared with corresponding parameters in the control.

The test method of application and the test species Daphnia magna are recommended by the test guidelines.

The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.

The test was performed according to the following guidelines, compliant with GLP:

- EU Method C.20.: "Daphnia magna Reproduction Test", Commission Regulation (EC) No 735/2017, Appendix 8, Official Journal of the European Union (EN) L 112 Volume 60, dated April 28, 2017

- OECD Guideline for Testing of Chemicals, No. 211: "Daphnia magna Reproduction Test", adopted October 02, 2012

- OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures", December 15, 2000

- SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414 for assessment of validity of analytical data.

This study encompassed 1 test item concentration of 0.16 µg test item/L (in 100 µL Dimethylformamide (DMF)/L and 10 µL Tween 80/L), a solvent control (100 µL DMF/L and 10 µL Tween 80/L) and a control, with 4 replicates, each containing 10 Daphnia in separate cages. Tween 80 below the critical micelle concentration (CMC) had to be added to prevent daphnids from being trapped on the surface in the solvent control as well as the treatment. This phenomenon was not observed in the control without solvent and thus seems to be related to the presence of DMF (100 µL/L) in these groups. Endpoints assessed were mortality of adults and number of offspring per survived parent (no symptoms of intoxication observed).

The quantification of the test item Matrilox LP101M in the test samples was performed using liquid chromatography (HPLC) with MS/MS detection. All four replicates were regularly assessed at day 0, 7, 14 and 20. Further, sampling at day 4 before introduction of animals confirmed successful equilibration of the final test item concentration for all four replicates of the limit concentration.

In the test media, 97 % of the nominal test concentration was found (mean values of nominal 0.16 µg/L, corrected for the recoveries of 75 % in the fortified samples, correction factor 1.33) throughout the test (arithmetic mean value). Correct dosing of the test item could be demonstrated. Accordingly, all reported results refer to nominal values since the concentrations of the test item were within ± 20 % of the nominal concentrations during the test.

Biological results:

At the saturation level in test medium (corresponding to the nominal limit concentration of 0.16 µg test item /L) not any toxic effect of the test item relative to controls could be observed:

The 21 day NOEC was determined to be ≥ 0.16 µg test item/L for the reproduction and ≥ 0.16 µg test item/L for the mortality of the adult test animals.

The LOEC / EC50 for the reproduction and mortality of adults was > 0.16 µg test item/L.