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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2017-11-20 to 2018-03-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: all concentrations were sampled but only the undiluted test medium (equilibrated test medium with a loading rate of 100 mg/L) and the dilution 1:4 and 1:2 were analysed. The samples of the dilutions 1:8 and 1:16 were not analyzed since these concentrations were below the 48-hour NOEC determined in this test and were therefore not relevant for the interpretation of the biological results.
- Sampling method: For the determination of the actual test item concentrations, duplicate samples were taken from each test concentration and the control at the start and at the end of the test. For sampling from the aged test media, the contents of the respective replicates were combined prior to sampling.
- Sample storage conditions before analysis: All samples were stored frozen (at -20 ± 5 °C) immediately after sampling. Based on analytical pre-experiments for investigation of the storage stability, the test item was found to be stable in the test water under these storage conditions.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Pre-Experiment for Test Medium Preparation:
To determine the solubility of the poorly soluble test item (water solubility < 100 mg/L) and the optimum time period to reach equilibration between dissolved and undissolved test item within a reasonable time period, the following stirring pre-experiment was performed:
As the test item is a liquid with low water solubility (<100 mg/L), the slow-stirring method was applied for preparation of an equilibrated test item solution. The slow-stirring method is an alternative method for preparing equilibrated solutions for insoluble, liquid test items, which are suspected to pass filters during filtration process.
In this stirring experiment the test item was carefully applied onto the surface of the test water in the almost completely filled stirring vessel at a loading rate of 100 mg/L without mixing the test item into the water column. Then, the stirring vessel was tightly sealed with glass stoppers. A small headspace in the mixing vessel had to be included for practical reasons as the test item was floating on the water surface.
Then, slow stirring was started and samples were taken after 24, 48, 72 and 96 hours. The samples were carefully harvested from the lower part of the water column through a tap at the bottom of the stirring vessel and as a precaution, the medium was checked by the Tyndall effect for the presence of undissolved test item particles. The upper part in the mixing vessel of the test medium with undissolved test item floating on the surface remained in the stirring vessel and was not used.
These results showed that the maximum concentration of dissolved test item in test water was reached after a stirring period of 96 hours. Additionally, an aliquot of the test medium after 24 hours stirring period (0.87 mg/L test item) was incubated for 48 hours at room temperature in an open and in a closed system. The test item concentrations in these test media after incubation were 0.012 and 0.83 mg/L, in the open and closed system, respectively. This demonstrates the stability of that test item concentration at room temperature overs 48 hours (in a closed system) and the volatility of the test item. These results suggested a closed system for the range-finding test.

At the start of the test the undiluted equilibrated test medium with a loading rate of 100 mg/L, was prepared following the slow-stirring method (see above) with a stirring period of 96 hours at room temperature in the dark. 289.4 μL of test item were carefully applied (pipetted) onto the surface of 2310 mL test water. This volume is equivalent to a loading rate of 100 mg/L, considering the density of the test item of 0.798 g/cm3 (at 20 °C). The undiluted equilibrated test medium was used as the highest test concentration, which was further diluted with test water to prepare the test media with the lower test concentrations.
Evidence of undissolved material (e.g. precipitate, surface film, etc.): All solutions were clear with no evidence of undissolved test item. No auxiliary solvent or emulsifier was used.
The test media were prepared just before the start of the test (i.e., introduction of the daphnids to the test media).
The preparation of the test media was based on the OECD Guidance Document No. 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna Straus
- Source/Age of parental stock: a clone of this species (originally from the Daphnia Collection of the University of Basel/Switzerland in 2015) is successfully bred in IES Ltd Laboratories.
- Feeding during test: no
- Breeding conditions:
- Food type/Amount/Frequency: fed three times a week with an algal suspension of the green algae Desmodesmus subspicatus, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen/Germany) and cultivated at IES Ltd Laboratories under standardized conditions or a mixture of this algal suspension and a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-GmbH, 49324 Melle/Germany).
- Culture medium and conditions: reconstituted water of the quality identical to the water quality used in the tests (with respect to pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests:
Reconstituted test water (ISO Test water) according to OECD Guideline No. 202. The ratio of Ca:Mg and Na:K was 4:1 and 10:1, respectively, based on molarity. The test water was aerated prior to the start of the study until oxygen saturation was reached. During the test period, the test water was not aerated.

ACCLIMATION: not needed since water quality is the same between breeding and testing

At the start of the test, the organisms used in the test were 6-24 hours old and were not first brood progeny.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Hardness:
250 mg/L as CaCO3
Test temperature:
The test was performed in a temperature-controlled room with continuous monitoring of the room temperature. The water temperature was maintained at 21 °C.
pH:
7.9-8.0
Dissolved oxygen:
8.5-8.6 mg/L
Nominal and measured concentrations:
Nominal concentrations: undiluted test media (loading rate 100 mg/L), dilutions 1:2, 1:4, 1:8, 1:16
Measured concentrations: 1.7, 0.89, 0.43 mg/L (Mean Measured Concentrations of the Test Item during the 48-Hour Test Period). The samples of the dilutions 1:8 and 1:16 were not analyzed since these concentrations were below the 48-hour NOEC.
Details on test conditions:
TEST SYSTEM
- Test vessel: in glass vessels completely filled (without headspace) with 60 mL test medium and tightly sealed with glass stoppers to avoid losses of the volatile substance by evaporation (closed system).
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: The volume of test medium provided for each daphnid was 12 mL (60 mL per replicate).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water (ISO Test water) according to OECD Guideline No. 202 was used in the study. It consisted of analytical grade salts dissolved in purified water at the following nominal concentrations:
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
Water Hardness 2.5 mmol/L; 250 as CaCO3 mg/L
Alkalinity 0.8 mmol/L
The ratio of Ca:Mg and Na:K was 4:1 and 10:1, respectively, based on molarity. The test water was aerated prior to the start of the study until oxygen saturation was reached. During the test period, the test water was not aerated.

- Culture medium different from test medium: no
- Intervals of water quality measurement: pH, dissolved oxygen were measured at T0 and 48H. Temperature-controlled room with continuous monitoring of the room temperature.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16-hour light to 8-hour dark cycle with a 30-minute transition period.
- Light intensity: between 15 and 17 μmol m-2 s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The immobility of the daphnids was determined by visual inspection after 24 and 48 hours of exposure. Those daphnids not able to swim within 15 seconds after gentle agitation of the test vessel were considered to be immobilized. Observations were also performed for other non-lethal effects (e.g. discoloration, surface trapping, reduced swimming etc.).

VEHICLE CONTROL PERFORMED: not appropriate

RANGE-FINDING STUDY:
Two replicates with 5 daphnids each per treatment were tested in parallel with a control.
The test concentrations were the undiluted equilibrated test medium with a loading rate of 100 mg/L and the dilutions 1:5 and 1:25. For preparation of the undiluted equilibrated test medium, the slow-stirring method was applied. The stirring time was 96 hours based on the results of the stirring pre-experiment. The undiluted equilibrated test medium was used in a series of dilutions with test water for preparation of the lower concentrations of test media (dilutions 1:5 and 1:25).
The analytical results at the end of the test were 59, 94 and 99 % of the initially measured values, respectively. The results show, that the test item is stable over a period of 48 hours at the two highest test concentrations.
Results: undiluted test media: 60% immobilisation at 24H, 100% at 48H. 1:5: 0% at 24 and 48H. 1:25: 0% at 24 and 48H.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate is tested as a positive control twice a year.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.91 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 0.78-1.1 mg/L
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.43 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: =EC0
Key result
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
1.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: reduced swimming acitvity was noted in the test solutions containing test substance (see details in results).
- Mortality and other adverse effects of control: in the control no daphnids showed immobilization or other signs of disease or stress (e.g., discoloration, surface trapping, reduced swimming etc.).
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no, No remarkable observations were made concerning the appearance of the test media. All test media remained clear solutions throughout the test.
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
The result of the latest positive control test in October 2017 (24-hour EC50: 1.3 mg/L) showed that the sensitivity of the test organisms was within the range given by the guideline (24-hour EC50: 0.60-2.1 mg/L).
Reported statistics and error estimates:
The 24-hour EC50 of the test item could not be calculated, because none of the responses exceeded 50%. The EC 50 was, therefore, also determined directly from the raw data.
The 48-hour EC50 and the 95 % confidence limits were calculated by Weibull Analysis.
The lowest nominal concentrations corresponding to the dilutions of 1:8 and 1:16 were not taken into account at the Weibull Analysis, because they were below the determined 48-hour NOEC and, thus, not analyzed.
Statistical analysis was performed using ToxRat Professional®.
The NOEC, EC0 and EC100 were determined directly from the Raw Data.

Any other information on results incl. tables

The measured concentrations of the test item TRI-N-HEXYLAMINE in the undiluted test media and the dilutions 1:2 and 1:4 at the start and end of the test demonstrate the correct preparation of the test media, i.e. the spacing factor of 2 between the different test concentrations was met. At the end of the test, 86 to 94 % of the initially measured values were found, demonstrating that the test item concentrations were sufficiently stable in the test media over the test period of 48 hours.

The mean measured test item concentration during the test period of 48 hours were calculated as geometric means:

 

Treatment / Dilution#

Analytically Measured Test Item Concentrations [mg/L]

Mean Measured Concentrations of the Test Item during the
48-Hour Test Period

[mg/L]

Day 0

Day 2

1:4

0.464

0.396

0.43

1:2

0.921

0.852

0.89

Undiluted Test Medium*

1.780

1.679

1.7

 

*:          Equilibrated test medium with a loading rate of 100 mg/L.

#:          Dilutions of the equilibrated test medium with a loading rate of 100 mg/L.

 

During the first 24 hours of the test, no immobilized test organisms were determined in the control and up to and including the mean measured concentration of 0.43 mg/L. At the next higher concentration, 0.89 mg/L, 5 % of the daphnids were found to be immobile and two test organisms were found to have reduced swimming activity. At the highest mean measured concentration of 1.7 mg/L, 20 % of the daphnids were found to be immobile and seven daphnids showed reduced swimming activity.

After 48 hours of exposure, no immobilized test organisms were determined in the control and up to and including the mean measured concentration of 0.43 mg/L. At the next higher test concentration, 0.89 mg/L, 10 test organisms were found to be immobile, corresponding to an immobilization rate of 50 %. In addition at 0.89mg/L, all surviving daphnids showed a reduced swimming activity. At the highest test concentration of 1.7 mg/L, all daphnids were found to be immobile.

 

Effect of TRI-N-HEXYLAMINE on the Mobility of Daphnia magna:

Treatment / Dilution#

Mean Measured
Concentration

No. of
Daphnids/ Replicate

Immobilized
Daphnids after 24 Hours

Immobilized
Daphnids after 48 Hours

 

[mg/L]

 

No.

[%]

No.

[%]

Control

---

5

0

0

0

0

5

0

0

5

0

0

5

0

0

1:16

n.a.

5

0

0

0

0

5

0

0

5

0

0

5

0

0

1:8

n.a.

5

0

0

0

0

5

0

0

5

0

0

5

0

0

1:4

0.43

5

0

0

0

0

5

0

0

5

0

0

5

0

0

1:2

0.89

5

0 (1F)

5

2 (3F)

50

5

1

2 (3F)

5

0

3 (2F)

5

0 (1F)

3 (2F)

Undiluted Test Medium*

1.7

5

2 (1F)

20

5

100

5

1 (2F)

5

5

0 (2F)

5

5

1 (2F)

5

 

*: Equilibrated test medium with a loading rate of 100 mg/L.

#: Dilutions of the equilibrated test medium with a loading rate of 100 mg/L.

n.a.:           Not analyzed.

Values in parenthesis:    Number of mobile test animals with adverse effect

A:       daphnids trapped at the water surface

B:       daphnids sticking together

C:       antennae sticking together

D:      daphnids discolored/pale

E:       spina stuck

F:       reduced swimming activity

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
in the control no daphnids showed immobilization or other signs of disease or stress. The dissolved oxygen concentration at the end of the test was ≥3 mg/L in all test vessels.
Conclusions:
The test item TRI-N-HEXYLAMINE had acute toxic effects on Daphnia magna in a 48-hour static test in a closed system. The biological test results (based on mean measured concentrations) were as follows:
48-hour EC50 : 0.91 (95 % Confidence Limits) (0.78 – 1.1) mg/L
48-hour EC0 and 48-hour NOEC: 0.43 mg/L
48-hour EC100: 1.7 mg/L
Executive summary:

The acute toxicity of the test item TRI-N-HEXYLAMINE to Daphnia magna was determined in a 48-hour static test according to the OECD Guideline for Testing of Chemicals, No. 202 (2004) and the Commission Regulation (EC) No. 440/2008, Part C.2 and following GLP. As the test item is a volatile substance, the test was performed using glass tubes completely filled (without headspace) with test medium that were tightly sealed with glass stoppers to avoid losses of test item by evaporation (closed system). A static test design, with no test medium renewal, was used.

As the test item is a liquid with low water solubility, the slowstirring method (to avoid formation of micro emulsions) was applied for preparation of a saturated test item solution. For preparation of the highest concentration of test medium, the test item with specific gravity of 0.798 was carefully applied (pipetted) onto the surface of the test water at a loading rate of

100 mg/L. Thereafter slow-stirring was applied for 96 hours in a closed vessel to reach a maximum concentration of dissolved test item in the test water. The stirring vessel was nearly completely filled (a small headspace had to be included as the test item was floating on the water surface) and tightly sealed with glass stoppers to avoid losses of the volatile test item during stirring.

After this treatment the lower part of the equilibrated test medium was carefully harvested from the stirring vessel through a tap at the bottom of the vessel. This equilibrated aqueous phase with a loading rate of 100 mg/L, containing dissolved test item only, was used as the highest concentration and was diluted with test water to obtain the dilutions 1:2 1:4, 1:8 and 1:16.

Additionally, a control (test water without test item) was tested in parallel.

The analytical results demonstrate the correct preparation of the test media, i.e. the spacing factor of 2 between the different test concentrations was met. At the end of the test, 86 to 94 % of the initially measured values were found, demonstrating that the test item concentrations were sufficiently stable in the test media over the test period of 48 hours.

A clear dose-response effect was observed after the exposure period of 48 hours: At the three lowest test concentrations no toxic effect was observed until test end. At the two highest test concentrations of 0.89 and 1.7 mg/L, 50 and 100 % of the daphnids were found to be immobile after 48 hours, respectively. The following data were obtained (based on mean measured concentrations):

48-hour EC50 : 0.91 (95 % Confidence Limits) (0.78 – 1.1) mg/L

48-hour EC0 and 48-hour NOEC: 0.43 mg/L

48-hour EC100:  1.7 mg/L

All the validity criteria were met.