Propane-1,2,3-triyl trinonan-1-oate

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-09-22 to 2018-04-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

adopted July 26, 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Biodegradability: Readily biodegradable, see IUCLID section 4.2.1;
Water solubility: 0.078 μg/L (Sonntag 2017), see IUCLID section 4.8;
Storage Conditions at Test Facility: At 20 ± 5 °C, in the dark.

Analytical monitoring:

yes

Details on sampling:

One sample from the freshly prepared stock solutions and one sample from each replicate (aquaria) of the test media of all test concentrations and the controls was taken prior to the initiation of the test. Afterwards once per week at day 0 (=start of the test), 3, 10, 17, 24 and 30 days post hatch, one sample from each replicate (aquaria) from the test media of all test concentrations and the controls was taken. In addition, one sample from the freshly prepared stock solutions was taken at day 0 (=start of the test), 4, 11, 18 and 25 days post hatch. All test medium samples were taken from the approximate centre of the aquaria. One aliquot (apart from stock solution samples in organic solvent) of the samples was diluted by a factor of 2 with acetonitrile. Another aliquot remained undiluted until sample preparation, which was performed directly after sampling.
Storage: All undiluted samples were extracted with the organic solvent n-hexane directly after
sampling. An aliquot of the n-hexane extracts as well as all samples diluted with acetonitrile were
stored in a freezer (≤ - 20 °C), protected from light until analysis was performed. Afterwards the s
amples 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 each of the un
diluted test media samples from all test concentrations, and in each of the undiluted control samples, from all sampling times. In addition, the test item was analysed in the stock solution samples taken at day 0 and 4 days post hatch. The samples diluted with acetonitrile were not analysed.
Details on analytical methods
Analytical Standard:
The test item was used to prepare the stock solution and the standard solutions.
Standard Solutions used for the Quantification
Stock Solution:
The test item was used to prepare a stock solution. At the first analysis (equilibration phase), 49.01 mg of test item was dissolved in 50 mL acetonitrile (with 1 minute ultrasonication) to obtain a stock solution of approximately 1 g test item /L. At the second analysis (test start and 3 days post hatch samples), 50.79 mg of test item was dissolved in 50 mL acetonitrile (with 2 minutes ultrasonication) to obtain a stock solution of approximately 1 g test item /L. At the third analysis (10 and 17 days post hatch samples), 51.39 mg of test item was dissolved in 50 mL acetonitrile (with 3 minutes ultrasonication) to obtain a stock solution of approximately 1 g test item /L. At the fourth analysis (24 days post hatch and test end samples), 50.04 mg of test item was dissolved in 50 mL acetonitrile (with 2 minutes ultrasonication) to obtain a stock solution of approximately 1 g test item /L. At the fifth analysis (re-analysis of selected samples), 49.75 mg of test item was dissolved in 50 mL acetonitrile (with 3 minutes ultrasonication) to obtain a stock solution of approximately 1 g test item /L.
Standard Solutions:
Appropriate amounts of the stock solutions were diluted with acetonitrile to obtain intermediate solutions of 1 µg test item /L. Appropriate amounts of this intermediate solution were diluted further with acetonitrile / test water (1/1, v/v) to obtain standard solutions in the range from 0.02 (first analysis), 0.05 (second and third) or 0.025 (fourth and fifth analysis) to 0.45 µg test item /L (upper range used for all analyses). Exact data were documented in the raw data.

Analytical Samples
Fortified Samples:
Fortified samples were prepared on each day of analysis. Approximately 50 mg of the test item were dissolved (1 - 3 minutes ultrasonication) in 50 mL acetonitrile to obtain a stock solution of approximately 1 g test item/L. Ten independent stock solutions were prepared. Appropriate amounts of these stock solutions were diluted with acetonitrile to obtain intermediate solutions of 2 (fourth and fifth analysis) or 4 (first, second and third analysis) and 15 µg test item/L (all analyses). Appropriate amounts of these intermediate solutions were diluted with test water to obtain fortified samples at a level of 0.02 (fourth and fifth analysis) or 0.04 (first, second and third analysis) and 0.15 µg test item/L (all analyses). Exact values were documented in the raw data.
Sample Preparation Procedure Sample Preparation:
A) Biological treatment samples and control samples:
Sample preparation was performed directly after sampling. The samples were shaken well and 5 mL n-Hexane was added to a sample volume of 25 mL present in 40 mL glass flasks. Extraction was performed for 15 – 20 minutes in an overhead shaker followed by the removal of the n-Hexane phase with a pipette. The extraction was performed twice and the n-Hexane extracts were unified.
An aliquot of the unified extract was stored in a freezer (≤ - 20 °C), in case a re-analysis were necessary.
N-Hexane extracts taken from the freezer were adapted to room temperature before sample preparation. Afterwards, 1 mL of the n-Hexane extract was added to a 2 mL vial and was evaporated under a nitrogen stream. After evaporation, the test item was dissolved in 1 mL test water/acetonitrile (1/1, v/v) by intense vortexing.
B) Stock solutions in DMF:
The samples were thawed to room temperature, ultrasonicated for 1 minute and shaken well. The
samples were diluted with acetonitrile by factor 1000. They were diluted further with test water/acetonitrile (1/1, v/v) to match the calibration range.
C) Fortified samples and analytical blank control samples:
The samples were shaken well and 5 mL n-Hexane was added to a sample volume of 25 mL present in 40 mL glass flasks. Extraction was performed for 15 – 20 minutes in an overhead shaker followed by the removal of the n-Hexane phase with a pipette. The extraction was performed twice and the n-Hexane extracts were unified. Afterwards, 1 mL of the n-Hexane extract was added to a 2 mL vial and was evaporated under a nitrogen stream. After evaporation, the test item was dissolved in 1 mL test water/acetonitrile (1/1, v/v) by intense vortexing.
LC-MS/MS-Conditions:
please see section "Any other information on materials and methods incl. tables".

Result Evaluation
Identification:
The identity of the analyte was confirmed by the high specificity of the mass transitions and by
comparison of the retention time with the retention time of a standard solution prepared from the test item.
Quantification:
Samples were quantified by measuring the peak area with reference to the calibration curve. The latter was obtained by correlation of peak area of the standard solutions to their corresponding concentration. The correlation was performed using a linear regression function given by equation (1):
y = a * x + b (1)
where
y: peak area
x: concentration of analyte
a: slope
b: y-axis intercept
Calculated Concentration:
The concentration of the analyte in the treatment samples and in the control sample was calculated by equation (2):
c = x * d (2)
where
c: concentration of analyte in original sample
x: concentration of analyte in the analysed sample (calculated in equation (1))
d: dilution factor
Recovery Rate:
The recovery rate (% of nominal) in a sample was calculated by equation (3):
% of nominal = (c/cnoma ) * 100 % (3)
where
c: concentration of analyte in original sample, cf. equation (2)
cnoma: nominal concentration of analyte
Limit of Detection: T
The Limit of Detection (LOD) of the method is defined as the lowest concentration having a peak
height equivalent to or better than three times the baseline noise.
Limit of Quantification:
The Limit of Quantification (LOQ) was determined as the lowest fortification level at which an
acceptable mean recovery (70 to 110% of nominal) with a relative standard deviation (RSD) ≤ 20% was obtained.

Vehicle:

yes

Remarks:

N,N-Dimethylformamide (DMF) at 20 µL/L

Details on test solutions:

Test Concentrations (nominal) definitive test:
0.12, 0.08, 0.05 μg test item/L (each in 20 μL DMF/L);
Controls:
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 the solvent 20 μL DMF/L but without t
he test item.
Dosage of Test Item:
A concentrated stock solution of 300 mg test item/L DMF was prepared by dissolving:
15.0 mg test item in 50 mL DMF or
15.4 mg test item in 51.3 mL DMF or
16.0 mg test item in 53.3 mL DMF
by intensive stirring for 1 - 2 min.
Stock solutions for each test concentration were prepared with 6000, 4000 and 2500 μg test item/L
DMF. In order to reach the requested concentrations of 0.12, 0.08, 0.05 μg test item/L the stock sol
utions of test item were dosed with 0.278 μL/min (± 10%) and mixed with 13.9 mL/min (± 10%) of
test water.
For the control 13.9 mL/min (± 10%) of test water was carried.
For the solvent control 0.278 μL/min (± 10%) DMF were mixed with 13.9 mL/min (± 10%) of test water.
The dosage of the test item stock solution and solvent was performed with a syringe pump. The
accuracy of the dosage was checked before equilibration phase and after the exposure period (en
d of biological part). Accordingly, over all groups and replicates, a minimum flow of 0.254 µL/min (
91% of nominal) and a maximum flow of 0.278 µL/min (100% of nominal) could be demonstrated and
such, correct dosing could be confirmed. An operation checkout of the syringe pumps was perfor
med twice a week during exposure phase.
The dosage of the test water was performed with a tube pump. The accuracy of the dosage was
checked 9, 6, 2 and 1 days prior the initiation of the experiment. After insertion of the eggs checks
on accuracy were performed twice a week. Accordingly, over all groups and replicates, a minimum
flow of 13 mL/min (94% of nominal) and a maximum flow of 15 mL/min (108% of nominal) could be
demonstrated and such, correct dilution water flow could be confirmed.
The stock solutions and the test water were pumped in mixing vessels (Erlenmeyer glass flask, one
per replicate) with a constant flow rate by syringe pumps (test item stock solutions or flexible-tube
pumps (test water), respectively. In these mixing vessels, the application stock solutions and the test
water were continuously mixed using a magnetic stirrer. Nominal test concentrations of 0.12, 0.08, a
nd 0.05 μg test item/L did result. The mixing vessels and the aquaria were connected by a tube (Poly
tetrafluoroethylene (PTFE)).
The stock solutions were renewed every 3 - 4 days.
Prior to the initiation of the test, the dosing system was calibrated through the use of appropriate
analysis techniques (this part was not performed according to the GLP-Regulations and is excluded
from
the Statement of Compliance in the final report, but the raw data of these tests are archived under the
project number of the present study).
Appearance of the Test Item in Test Medium:
The appearance of the test item in the test medium was observed once every day in all test concent
rations.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Species: Zebrafish (Danio rerio)
Age: Freshly fertilised eggs (before beginning of gastrulation)
Origin: The test eggs were obtained from the brood stock of the ibacon GmbH

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

34 d

Hardness:

124.6 to 160.2 mg CaCO3/L

Test temperature:

25.1 to 26.4 °C

pH:

7.6 to 8.0

Dissolved oxygen:

96 to 102 % of the air saturation value

Conductivity:

Deionised water (conductivity <= 10 µS/cm) was used for preparation of the test medium (ISO
medium according to OECD 203).

Nominal and measured concentrations:

0.12, 0.08, 0.05 μg test item/L (each in 20 μL DMF/L), corresponding to the following mean measured
concentrations of the test item, corrected for recovery of fortified samples:
0.0600, 0.0328 and 0.0243 μg test item/L (each in 20 μL DMF/L).
Based on results from the preliminary test and the determined water solubility of 0.078 µg/L, no t
oxicity of the test item up to the saturation concentration in water was expected. Therefore, it was
decided that an extended limit test as foreseen according to OECD 210, paragraph 22 would be most
appropriate in this case. Using a lower spacing factor of 1.5 and testing three test item concentrati
ons resulted in actually applied nominal test item concentrations of 0.12 µg test item/L (water solu
bility times a safety factor of 1.5 as a reasonable compromise between trying to avoid a relevant no
n-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), 0.08, and 0.05 µg test item/L.
While reducing the normally required 5 test item concentrations to three, analytical monitoring was
extended beyond the minimum required according to OECD 210 (weekly, at least in one replicate per
concentration level), in that regularly all four replicates per concentration level were analysed. For
details on considerations regarding test design see IUCLID section "Any other information on materi
als and methods incl. tables".

Details on test conditions:

Plese see IUCLID section "Any other information on materials and methods incl. tables".

Reference substance (positive control):

no

Key result

Duration:

34 d

Dose descriptor:

LOEC

Effect conc.:

> 0.06 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks on result:

other: corresponding to the saturation level in test medium

Key result

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.06 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks on result:

other: corresponding to the saturation level in test medium

Key result

Duration:

34 d

Dose descriptor:

LOEC

Effect conc.:

> 0.06 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

length

Remarks on result:

other: corresponding to the saturation level in test medium

Key result

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.06 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

length

Remarks on result:

other: corresponding to the saturation level in test medium

Key result

Duration:

34 d

Dose descriptor:

LOEC

Effect conc.:

> 0.06 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: corresponding to the saturation level in test medium

Key result

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.06 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: corresponding to the saturation level in test medium

Key result

Duration:

34 d

Dose descriptor:

LOEC

Effect conc.:

> 0.06 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Remarks on result:

other: corresponding to the saturation level in test medium

Key result

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.06 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Remarks on result:

other: corresponding to the saturation level in test medium

Details on results:

Up to the saturation level in test medium (corresponding to the mean measured concentration of
0.060 µg test item /L) not any toxic effect of the test item relative to controls could be observed. While analytically determined mean measured concentration over all replicates for the highest nominal concentration of 0.12 µg/L test item was somewhat below the determined solubility in test medium (0.078 µg/L), the determined arithmetic mean measured concentration for replicate 3 over all days (0.072 µg/L) corresponds to 92% of the water solubility, while arithmetic mean measured concentration for replicate 3 up to and including day 24 post hatch (28 days of exposure) (0.084 µg/L) is even slightly above the saturation level of the test item in ISO medium (107% of the water solubility). No distinct differences to other replicates of this test concentration were obvious, neither for dry / wet weight or fish length, nor for hatching success or post hatch survival. This corroborates that up to the saturation level in water, no effects of the test item on fish early life stages occurred.
For further details, please see IUCLID sections "Any other information on results incl. tables" as well
as "Overall remarks, attachments".

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Please see IUCLID section "Any other information on materials and methods incl. tables".

Validity Criteria of the Study
Control Survival:      
In the control the survival of fertilised eggs was 100% being > 70 % (validity criterion). The post hatch
survival was 96 % being > 75 % (validity criterion). The overall mortality (including coagulated eggs, dead
embryos and dead larvae was 4 %.
Thus the validity criterion for survival in the control at the end of the study was met.
Water Temperature:
The water temperature differed not more than ±1.5°C between the test vessels or between the days at
any time during the test.
Oxygen Concentration:      
The dissolved oxygen concentration in the test media did not fall below 60% of air saturation value
during the test.
Analytical measurement:      
The test concentrations were verified at test start in all replicates (aquaria) and at least once a week
afterwards throughout the test until test end. Since the measured concentrations did not remain within
80-120% of the nominal concentration, the effect concentrations were expressed as arithmetic mean
concentrations.

 Biological Results       
 Embryonic Stage at Test Start:      
8 -16 cell stage;

 Number of Coagulated Eggs:      
No eggs coagulated in any treatment group.

 Hatching Time and Success:      
First larvae hatched from eggs 2 days after insertion in the control and all test item treatment groups. >
90 % of eggs were hatched at day 4 after insertion (start of post hatch period). Hatching was completed
5 days after insertion of eggs in the control and all test item treatment groups. The LOEC for hatchability
was determined to be > 0.0600 µg test item/L, and the NOEC was ≥ 0.0600 µg test item/L (100%
hatching success in all control and treatment groups).

 Mortality and Sublethal effects:  

No test item related sublethal effects occurred. At 0.0328 µg test item /L one fish tumbled and one fish
was found mainly on the bottom. However, these abnormalities were in the range of biological variance.
During the test several larvae died in all treatment groups and the controls. The survival in all treatment
groups was comparable to the pooled control and therefore within the biological variance. The calculated LOEC for post hatch survival was determined to be > 0.0600 µg test item/L, and the NOEC was ≥ 0.0600 µg test item/L.

Length:      
Mean total length for the control, solvent control and all test item concentration fish 30 days post hatch
was between 11.7 and 12.95 mm. The calculated LOEC for body length was determined to be > 0.0600
µg test item/L, and the NOEC was ≥ 0.0600 µg test item/L.
Concerning the mean length, no distinct differences were observed when comparing fish exposed
to the three test item concentrations with fish from the pooled control. In the highest nominal test
concentration of 0.0600 µg test item/L, fish of replicate 3 had the highest average length although
they were exposed to the highest arithmetic mean concentration compared to the other 3 replicates
within this test concentration. For details on biological results, see Table B-1, IUCLID section "Overall
remarks, attachments"; for details on analytical data see Table 2 below (analytical summary) and
Table-A-1, IUCLID section "Overall remarks, attachments" for analytical details on the highest test item
concentration of nominal 0.12 µg/L.

Wet Weight:      
Mean wet weight per fish for the control was 13.3 mg; the mean wet weight per fish in the solvent control
was 14.6 mg. The mean wet weight of larvae in the test item treatment groups ranged between 14.9 mg
and 15.5 mg. The calculated LOEC for body wet weight was determined to be > 0.0600 µg test item/
L, and the NOEC was ≥ 0.0600 µg test item/L.
Comparing the mean wet weight of fish exposed to the three test item concentration with fish from
the negative and the solvent control, no distinct differences were obvious. In the highest nominal
test concentration of 0.0600 µg test item/L, fish of replicate 3 had the highest average wet weight
although they were exposed to the highest arithmetic mean concentration compared to the other 3
replicates within this test concentration. For details on biological results, see Table B-1, IUCLID section
"Overall remarks, attachments"; for details on analytical data see Table 2 below (analytical summary)
and Table-A-1, IUCLID section "Overall remarks, attachments" for analytical details on the highest test
item concentration of nominal 0.12 µg/L.

Dry Weight:      
Mean dry weight per fish for the control was 2.8 mg; the mean dry weight for fish in the solvent control
was 3.2 mg. The mean wet weight of larvae in the test item treatment groups ranged between 3.2 and
3.4 mg. The calculated LOEC for body dry weight was determined to be > 0.0600 µg test item/L, and
the NOEC was ≥ 0.0600 µg test item/L.
Concerning the mean dry weight, also no distinct differences were observed between fish exposed
to the three test item concentrations and fish from the pooled control. In the highest nominal test
concentration of 0.0600 µg test item/L, fish of replicate 3 had the second highest average dry weight
although they were exposed to the highest arithmetic mean concentration compared to the other three
replicates within this test concentration. For details on biological results, see Table B-1, IUCLID section
"Overall remarks, attachments"; for details on analytical data see Table 2 below (analytical summary)
and Table-A-1, IUCLID section "Overall remarks, attachments" for analytical details on the highest test
item concentration of nominal 0.12 µg/L.

 Appearance of the Test Item in Test Medium:      
There were no remarkable observations.

 Analytical Results
 Limit of Detection:
0.004 μg test item/L;
 Limit of Quantification:
0.05 μg test item/L after enrichment by factor 2.5 (corresponding to fortification level of nominal 0.02
μg test item/L); Mean recovery: 103% (n = 4, RSD 6%, see Table A-3)
 Mean Recovery in the Test
Samples:
39% (n = 70, RSD 60%); for details on recovery for the highest nominal test item concentration of 0.12
µg/L, see Table A-1, IUCLID section "Overall remarks, attachments".
Mean Recovery in the DMF stock solutions:
81% (n = 6, RSD 21%

 Validity Criteria of the Analytical Part
According to the study plan, the method was validated according to GLP based on the criteria set forth
by SANCO/3029 (SANCO/3029/99 rev.4 11/07/00).
 Specificity:
No significant (< 30%) interference of total peak area for the target analyte was found.
 Linearity:
Calibration Range:
0.02 – 0.45 μg test item/L (first analysis)
0.05 – 0.45 μg test item/L (second and third analysis)
0.035 – 0.45 μg test item/L (fourth analysis)
0.025 – 0.35 μg test item/L (fifth analysis)
Linearity of Response:
Correlation of peak area of different standard solutions with their corresponding concentrations, using
a linear regression.
Regression Coefficients: r = 0.9936 (at least)
Example of Calibration Curve:
y = 1158390 * x – 15401
 Accuracy and Precision:
Mean Recovery Rates in the Fortified Samples: 84% (n = 17, RSD 16%). The values found for the precision
(RSD) and for the accuracy (mean recovery rate) are acceptable (for details see Table A-3, IUCLID section
"Overall remarks, attachments").
Due to recovery values of 84% for the applied method, final arithmetic mean concentrations of the test
item concentrations were corrected by factor 1.19 (100%/84%) to accurately reflect the performance
of the applied method and to avoid underestimation of the determined test concentrations (see Table
2 above).
 Conclusion: The validity criteria for the analytical method have been met.

 Discussion      
 Analytical results:      
The test item recoveries in all test concentrations showed a high fluctuation after test start with
a decreasing tendency towards test end (please see Table A-1, IUCLID section "Overall remarks,
attachments" for details on highest nominal concentration of 0.12 µg/L). Since the dosage system of
the test item worked correctly and almost nominal recoveries were determined in selected DMF stock
solutions (see Table A-2, IUCLID section "Overall remarks, attachments"), the application of the test item
was done appropriately.
The fluctuating concentrations are likely an effect of the test items high log Kow and its property of being
highly biodegradable. With continuing test duration, increasing amounts of biomass and microorganism
are existent in the aquaria due to growing fish, excrements and fish food. The high log Kow promotes
adsorption of the test item on biomass surface. Thus, the low recovery of the test item is likely due to
the presence of biomass and probably caused by adsorption as well as biodegradation losses.
These factors seemed to have a strong negative influence on analytical test item recovery despite of the
fact that all test item concentrations were renewed fivefold per day and the aquaria were kept as clean
as possible (as low surplus food as possible and regular cleaning of the aquaria) without stressing the
fish. Higher test water renewal rates would have been disadvantageous for fish hatching and survival,
especially at the presence of organic solvents such as DMF, which was shown in pre-experiments at the
testing facility. Therefore, the fivefold-renewal rate was a reasonable compromise to keep as much test
item as possible available on the one side and to have optimal conditions for the fish on the other side.
 Biological results:      
The arithmetic mean measured concentration of 0.0600 µg test item/L determined for the highest test
concentration of nominal 0.12 µg test item/L was slightly below the determined solubility of the test item
of 0.078 µg test item/L (Sonntag 2017). Nevertheless, analytical dose verification confirmed test item
availability for all test concentrations throughout the test, especially during the critical hatching phase
(please see details for highest nominal test item concentration of 0.12 µg/L in Table A-1, IUCLID section
"Overall remarks, attachments"). Emerging biological effects are not expected for concentrations above
0.0600 µg test item/L up to the determined solubility of 0.078 µg test item/L. This expectation is
underlined by replicate 3 of the highest test concentration of nominal 0.12 µg test item/L (see Table
2 above). Here, the highest arithmetic mean concentration of 0.0717 µg test item/L (0.0836 µg test
item/L until 24 days post hatch) was determined which corresponds to 92% of the test item solubility
(107% considering only determined concentrations up to and including 24 days post hatch). No distinct
differences to other replicates of this test concentration were obvious, neither for dry / wet weight or
fish length (see Table B-1, IUCLID section "Overall remarks, attachments"), nor for hatching success or
post hatch survival. Therefore, it seems reasonable to state that NOEC and LOEC values for hatching
success, mortality, wet and dry weight as well as length are very likely to be ≥ 0.078 µg test item/L
corresponding to the water solubility of the test item.

 Conclusion
 Up to the saturation level in test medium (corresponding to the mean measured concentration of
0.060 µg test item /L) not any toxic effect of the test item relative to controls could be observed:
The NOEC for hatching success was determined to be ≥ 0.0600 µg test item/L and the LOEC > 0.0600
µg test item/L.
Based on the test results, the NOEC for 30 days post hatch (DPH) survival was calculated  to be ≥ 0.0600
µg test item/L and the LOEC > 0.0600 µg test item/L.
For body length, the NOEC was calculated to be ≥ 0.0600 µg test item/L and the LOEC was calculated
to be > 0.0600 µg test item/L.
For body wet weight, the NOEC was calculated to be ≥ 0.0600 µg test item/L and the LOEC was calculated to be > 0.0600 µg test item/L.

For body dry weight, the NOEC was calculated to be ≥ 0.0600 µg test item/L and the LOEC was calculated
to be > 0.0600 µg test item/L.
No effect on mortality, body length, body dry and body wet weight occurred. Therefore no LCx or ECx
values were determined.
In the analytical part, prior to initiation of the exposure period and during the test, the test item
concentrations were determined in regular intervals to characterise exposure. All reported results refer
to arithmetic mean concentrations, since the test item concentrations were not within ± 20% of the
nominal concentrations during the test.

References:
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

Validity criteria fulfilled:

yes

Conclusions:

Up to the saturation level in test medium (corresponding to the mean measured concentration of
0.060 µg test item /L) not any toxic effect of the test item on Danio rerio early life stages could be observed relative to controls (OECD 210; GLP; 2018):
NOEC (30 d post hatch; overall survival, length, body dry/wet weight) >= saturation level in water;
LOEC (30 d post hatch; overall survival, length, body dry/wet weight) > saturation level in water.

Executive summary:

The purpose of this study was to evaluate the toxicity of the test item Matrilox LP101M (Trimethylolpropane trinonanoate) to the early-life stages of fish. For this purpose, fertilised eggs of Zebrafish (Danio rerio) were exposed in a flow-through test to aqueous test media containing the test item at various concentrations under defined conditions. The test duration was until 30 days after hatching. The recorded effects were mortality, hatching, growth, weight and sublethal effects of the fish.
The method used is recommended by the test guidelines, and also Zebrafish is one of the fish species recommended by the international test guidelines of the OECD and EC.
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:
-       OECD Guideline for Testing of Chemicals, Section 2, No. 210 "Fish, Early-life Stage Toxicity Test", adopted July 26, 2013.
-       OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures", December 15, 2000
This study encompassed 3 treatment groups (3 concentrations of the test item), a control and a solvent control, with 4 replicates each containing 25 freshly fertilized eggs. The eggs and larvae were observed
daily for sublethal effects and mortality. Dead larvae were removed at least once daily and discarded.
The test item concentrations in the test water taken two days before insertion of eggs, at start (insertion of eggs) and after 3, 10, 17, 24 and 30 days post hatch were analysed.
To determine the most appropriate test item concentrations for the definite test, a range-finding test was performed. Further, the solubility of the test item Matrilox LP101M in the test water (ISO-medium) was determined via a separately conducted GLP-study to a value of 0.078 µg/L (Sonntag, F, 2017). Based on results from the preliminary test and the determined water solubility of 0.078 µg/L, no toxicity of the test item up to the saturation concentration in water was expected. Both, OECD 210 as well as OECD No. 23 emphasize that it is highly important to conduct aquatic toxicity testing on truly dissolved test
item solutions only, i.e. up to the saturation level in water, but not beyond. Accordingly, the determined saturation level in ISO medium of 0.078 µg/L was used as the upper limit for aquatic toxicity testing on fish, multiplied with a safety factor of 1.5 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. This safety factor was based on close to nominal recoveries in the range-finding test. As such, an upper nominal test item
concentration of (rounded) 0.12 µg/L was derived. Testing five test item concentrations with a spacing factor of 3.2 as recommended according to OECD 210 would have resulted in testing concentrations pronouncedly below a nominal test item concentration of 0.05 µg/L and thus would have been out of
scope of the analytical quantification method used for verification of test item concentrations during the course of the study. Therefore, it was decided that an extended limit test as foreseen according to OECD 210, paragraph 22 would be most appropriate in this case. Using a lower spacing factor of 1.5 and three test item concentrations resulted in actually applied nominal test item concentrations of 0.12, 0.08, 0.05 µg test item/L. While reducing the normally required 5 test item concentrations to three, analytical monitoring was extended beyond the minimum required according to OECD 210 (weekly, at least in one replicate per concentration level), in that regularly all four replicates per concentration level
were analysed.
The quantification of the test item Matrilox LP101M was performed using liquid-liquid extraction with n-Hexane followed by the analysis via liquid chromatography (HPLC) with MS/MS detection. Two days before the start of the test (equilibration check of the flow through system), the mean test recoveries of the nominal test concentrations varied between 75 and 99% (all test concentrations considered). At the start of the test (day of egg insertion = DAI0), test item recoveries varied between 38 and 94% (all test concentrations considered). The further determined recoveries throughout the test until test end varied between 8 and 97% with a decreasing tendency towards test end. The fluctuating concentrations are likely an effect of the high log Kow of the test item and its property of being highly biodegradable. Due to deviation of analytically determined concentrations by more than ± 20% from nominal, all (no)effect concentrations are given based on arithmetic mean measured values.
 Results:
 Up to the saturation level in test medium (corresponding to the mean measured concentration of 0.060 µg test item /L) not any toxic effect of the test item relative to controls could be observed:
- The NOEC for hatching success was determined to be ≥ 0.0600 µg test item/L and the LOEC > 0.0600 µg test item/L.
- Based on the test results, the NOEC for 30 days post hatch (DPH) survival was calculated  to be ≥ 0.0600 µg test item/L and the LOEC > 0.0600 µg test item/L.
- For body length, the NOEC was calculated to be ≥ 0.0600 µg test item/L and the LOEC was calculated to be > 0.0600 µg test item/L.
- For body wet weight, the NOEC was calculated to be ≥ 0.0600 µg test item/L and the LOEC was calculated to be > 0.0600 µg test item/L.
- For body dry weight, the NOEC was calculated to be ≥ 0.0600 µg test item/L and the LOEC was calculated to be > 0.0600 µg test item/L.
No effect on mortality, body length, body dry and body wet weight occurred. Therefore no LCx or ECx values were determined.
While analytically determined mean measured concentration over all replicates for the highest nominal concentration (0.12 µg/L) of test item was somewhat below the determined solubility for the test
medium (0.078 µg/L), the determined arithmetic mean measured concentration for replicate 3 over all days (0.072 µg/L) corresponds to 92% of the water solubility, while arithmetic mean measured concentration for replicate 3 up to and including day 24 post hatch (28 days of exposure) (0.084 µg/ L) was even slightly above the saturation level of the test item in ISO medium (107% of the water solubility). The time span till day 24 post hatch covers the most sensitive develpmental phase. No distinct differences to other replicates of this test concentration were obvious, neither for dry / wet weight or fish length, nor for hatching success or post hatch survival. This corroborates that up to the
saturation level in water, no effects of the test item on fish early life stages occurred.

Description of key information

Up to the saturation level in test medium (corresponding to the mean measured concentration of 0.060 µg test item /L) not any toxic effect of the test item on Danio rerio early life stages could be

observed relative to controls (OECD 210; GLP; 2018):

NOEC (30 d post hatch; overall survival, length, body dry/wet weight) >= saturation level in water;

LOEC (30 d post hatch; overall survival, length, body dry/wet weight) > 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

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

> 0.06 µg/L

Additional information

One reliable key study is available for the endpoint long-term toxicity to fish.

The purpose of this study was to evaluate the toxicity of the test item Matrilox LP101M (Trimethylolpropane trinonanoate) to the early-life stages of fish. For this purpose, fertilised eggs of Zebrafish (Danio rerio) were exposed in a flow-through test to aqueous test media containing the test item at various concentrations under defined conditions. The test duration was until 30 days after hatching. The recorded effects were mortality, hatching, growth, weight and sublethal effects of the fish.

The method used is recommended by the test guidelines, and also Zebrafish is one of the fish species recommended by the international test guidelines of the OECD and EC.

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:

-       OECD Guideline for Testing of Chemicals, Section 2, No. 210 "Fish, Early-life Stage Toxicity Test", adopted July 26, 2013.

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

This study encompassed 3 treatment groups (3 concentrations of the test item), a control and a solvent control, with 4 replicates each containing 25 freshly fertilized eggs. The eggs and larvae were observed daily for sublethal effects and mortality. Dead larvae were removed at least once daily and discarded. The test item concentrations in the test water taken two days before insertion of eggs, at start (insertion of eggs) and after 3, 10, 17, 24 and 30 days post hatch were analysed.

To determine the most appropriate test item concentrations for the definite test, a range-finding test was performed. Further, the solubility of the test item Matrilox LP101M in the test water (ISO-medium) was determined via a separately conducted GLP-study to a value of 0.078 µg/L (Sonntag, F, 2017). Based on results from the preliminary test and the determined water solubility of 0.078 µg/L, no toxicity of the test item up to the saturation concentration in water was expected. Both, OECD 210 as well as OECD No. 23 emphasize that it is highly important to conduct aquatic toxicity testing on truly dissolved test item solutions only, i.e. up to the saturation level in water, but not beyond. Accordingly, the determined saturation level in ISO medium of 0.078 µg/L was used as the upper limit for aquatic toxicity testing on fish, multiplied with a safety factor of 1.5 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. This safety factor was based on close to nominal recoveries in the range-finding test. As such, an upper nominal test item concentration of (rounded) 0.12 µg/L was derived. Testing five test item concentrations with a spacing factor of 3.2 as recommended according to OECD 210 would have resulted in testing concentrations pronouncedly below a nominal test item concentration of 0.05 µg/L and thus would have been out of scope of the analytical quantification method used for verification of test item concentrations during the course of the study. Therefore, it was decided that an extended limit test as foreseen according to OECD 210, paragraph 22 would be most appropriate in this case. Using a lower spacing factor of 1.5 and three test item concentrations resulted in actually applied nominal test item concentrations of 0.12, 0.08, 0.05 µg test item/L. While reducing the normally required 5 test item concentrations to three, analytical monitoring was extended beyond the minimum required according to OECD 210 (weekly, at least in one replicate per concentration level), in that regularly all four replicates per concentration level were analysed.

The quantification of the test item Matrilox LP101M was performed using liquid-liquid extraction with n-Hexane followed by the analysis via liquid chromatography (HPLC) with MS/MS detection. Two days before the start of the test (equilibration check of the flow through system), the mean test recoveries of the nominal test concentrations varied between 75 and 99% (all test concentrations considered). At the start of the test (day of egg insertion = DAI0), test item recoveries varied between 38 and 94% (all test concentrations considered). The further determined recoveries throughout the test until test end varied between 8 and 97% with a decreasing tendency towards test end. The fluctuating concentrations are likely an effect of the high log Kow of the test item and its property of being highly biodegradable. Due to deviation of analytically determined concentrations by more than ± 20% from nominal, all (no)effect concentrations are given based on arithmetic mean measured values.

Results:

Up to the saturation level in test medium (corresponding to the mean measured concentration of 0.060 µg test item /L) not any toxic effect of the test item relative to controls could be observed:

- The NOEC for hatching success was determined to be ≥ 0.0600 µg test item/L and the LOEC > 0.0600 µg test item/L.

- Based on the test results, the NOEC for 30 days post hatch (DPH) survival was calculated  to be ≥ 0.0600 µg test item/L and the LOEC > 0.0600 µg test item/L.

- For body length, the NOEC was calculated to be ≥ 0.0600 µg test item/L and the LOEC was calculated to be > 0.0600 µg test item/L.

- For body wet weight, the NOEC was calculated to be ≥ 0.0600 µg test item/L and the LOEC was calculated to be > 0.0600 µg test item/L.

- For body dry weight, the NOEC was calculated to be ≥ 0.0600 µg test item/L and the LOEC was calculated to be > 0.0600 µg test item/L.

No effect on mortality, body length, body dry and body wet weight occurred. Therefore no LCx or ECx values were determined.

While analytically determined mean measured concentration over all replicates for the highest nominal concentration (0.12 µg/L) of test item was somewhat below the determined solubility for the test medium (0.078 µg/L), the determined arithmetic mean measured concentration for replicate 3 over all days (0.072 µg/L) corresponds to 92% of the water solubility, while arithmetic mean measured concentration for replicate 3 up to and including day 24 post hatch (28 days of exposure) (0.084 µg/L) was even slightly above the saturation level of the test item in ISO medium (107% of the water solubility). The time span till day 24 post hatch covers the most sensitive develpmental phase. No distinct differences to other replicates of this test concentration were obvious, neither for dry / wet weight or fish length, nor for hatching success or post hatch survival. This corroborates that up to the saturation level in water, no effects of the test item on fish early life stages occurred.