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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
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 two 24-hour test medium
renewal periods.
For sampling from the aged test media, the contents of the respective replicates were combined
prior to sampling.
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.
The concentrations of GR-86-6599 were analyzed in one of the duplicate test medium samples
taken from all sampling dates.
Vehicle:
no
Details on test solutions:
At the start of the renewal periods (on Day 0 and Day 1), the highest test medium with a loading rate of 50 mg/L was prepared following the slow-stirring method. For this, 125 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 50 mg/L, considering the relative density of the test item of 0.92. No auxiliary solvent or emulsifier was used.

The resulting test medium was slowly stirred for 48 hours at room temperature and in the dark. The mixing 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. After this treatment, stirring was stopped for 24 hours as a precaution to allow phase separation in case test item particles had been mixed into the water column during stirring. Following cessation of mixing and the period of settling, the lower aqueous and equilibrated phase was carefully separated from the non-dissolved upper test item phase through the tap at the bottom of the vessel. This equilibrated aqueous phase with a loading rate of 50 mg/L, containing dissolved test item only, was used as the highest test concentration and considered to represent the aqueous saturation concentration in test media of the test item. The highest test concentration was subsequently and diluted with test water to obtain the test media with the lower test concentrations. The dilutions prepared were, as follows, and were defined following a dose range finding study :

Undiluted Test Medium (loading rate of 50 mg/L)
Dilution 1 : 1.5
Dilution 1 : 2.3
Dilution 1 : 3.4
Dilution 1 : 5.1

Additionally, a control (test water only) was run in parallel.

The test media were prepared just before the start of the test (i.e., introduction of the daphnids to the test media) and prior to the test media renewal after 24 hours.

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 (species):
Daphnia magna
Details on test organisms:
The study was performed with young daphnids of the species Daphnia magna Straus. 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. The cultivation of the parental daphnids
is performed in 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.
During breeding, daphnids were generally 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-Werke,
49324 Melle/Germany).

At the start of the test, the organisms used in the test were 6 24 hours old and were not first brood progeny.
Test type:
semi-static
Water media type:
freshwater
Remarks:
Reconstituted test water according to ISO 6341.
Limit test:
no
Total exposure duration:
48 h
Hardness:
250 mg/L as CaCO3
Test temperature:
21°C
pH:
7.7 - 7.9
Dissolved oxygen:
At least 8.4 mg/L
Nominal and measured concentrations:
The results obtained for the concentrations of the test samples are presented in Table 7. The
concentrations determined in the freshly prepared test sample (day 0/0 hours) were 34.8 μg/L
(dilution 1:5.1), 43.8 μg/L (dilution 1:3.4), 72.5 μg/L (dilution 1:2.3), 99.9 μg/L (dilution 1:1.5)
and 135 μg/L (undiluted).
The concentrations determined in the test samples taken from the fresh test media
(day 1/0 hours) were 32.9 μg/L (dilution 1:5.1), 46.9 μg/L (dilution 1:3.4), 73.4 μg/L (dilution
1:2.3), 104 μg/L (dilution 1:1.5) and 151 μg/L (undiluted).
The concentrations determined in the test samples taken from the aged test media
(day 1 / 24 hours) were found to be 26.2 μg/L (dilution 1:5.1), 42.9 μg/L (dilution 1:3.4),
58.4 μg/L (dilution 1:2.3), 72.8 μg/L (dilution 1:1.5) and 99.2 μg/L (undiluted).
The concentrations determined in the test samples taken from the aged test media (day 2 /
24 hours) were found to be 25.8 μg/L (dilution 1:5.1), 48.4 μg/L (dilution 1:3.4), 58.7 μg/L
(dilution 1:2.3), 68.1 μg/L (dilution 1:1.5) and 114 μg/L (undiluted).
The results showed the correct preparation of the test samples. For most of the samples, the test
item concentration slightly decreased over the test period.
Details on test conditions:
The main test was performed in a closed system.

A semi-static main test with test medium renewal after 24 hours was chosen to further improve the maintenance of the test item concentrations during the test period of 48 hours. After 24 hours, the test organisms were transferred by wide-bore pipette in to clean test vessels with freshly prepared test medium of the corresponding concentration.

For each treatment, 20 daphnids were randomly distributed into four replicates of five daphnids each. The volume of test medium provided for each daphnid was 12 mL (60 mL per replicate). Thus, the requirement of the test guidelines for a minimum volume of 2 mL test medium per daphnid was fulfilled.

The test was performed in a temperature-controlled room with continuous monitoring of the room temperature. The water temperature was maintained at 21 °C.

The daphnids were not fed during the test.

A 16-hour light to 8-hour dark cycle with a 30-minute transition period was used. Light intensity during the light period was between 15 and 17 µmol m-2 s-1.
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 123 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Behaviour and appearance
Remarks on result:
other: This exposure concentration represents the maximum water saturation concentration of the test item.
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 123 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other:
Remarks:
This exposure concentration represents the maximum water saturation concentration of the test item
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
>= 123 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other:
Remarks:
This exposure concentration represents the maximum water saturation concentration of the test item
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 123 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other:
Remarks:
This exposure concentration represents the maximum water saturation concentration of the test item
Details on results:
The test is considered to be valid, as in the control no daphnids showed immobilization or other
signs of disease or stress (e.g., discolouration or unusual behaviour such as trapping at the
surface of water). Furthermore, the dissolved oxygen concentration at the end of the test was
≥3 mg/L in all test vessels.

After 48 hours of exposure, no immobilized test organisms were determined in the control and up to and including the highest test concentration of 123 µg/L, corresponding to the saturation concentration of the test item.

The 24 and 48-hour EC100 could not be determined due to the absence of toxicity of the test item.

No remarkable observations were made concerning the appearance of the test media. All test media remained clear solutions throughout the renewal periods.

As no toxic effect was observed, the 48-hour EC50, 48-hour EC100 were determined directly from the raw data to be >123 µg/L and the EC0 and NOEC to be ≥ 123 µg/L.
Results with reference substance (positive control):
Potassium Dichromate : EC50 (24 hour) = 1.0 mg/L

For evaluation of the quality of the daphnia clone and the experimental conditions, potassium dichromate is tested as a positive control twice a year. The result of the latest positive control test in October 2016 (24-hour EC50: 1.0 mg/L, IES Laboratories Study 20160278) 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).
Validity criteria fulfilled:
yes
Conclusions:
In conclusion the test item GR-86-6599 had no acute toxic effects on Daphnia magna in a 48-hour semi-static test up to the highest test concentration of 123 μg/L, corresponding to the saturation concentration of the test item.

48h EC50 > 123 μg/L
48h EC100 > 123 µg/L
48h EC0 ≥ 123 µg/L
48h NOEC ≥ 123 µg/L
Executive summary:

The acute toxicity of the test item GR-86-6599 to Daphnia magna was determined in a 48-hour

semi-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.

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 semi-static test design with a test medium

renewal after 24 hours was used.

As the test item is a liquid with low water solubility, the slow stirring method was applied for

preparation of a saturated test item solution. For preparation of the highest concentrated test

medium, the test item was carefully applied (pipetted) onto the surface of the test water at a

loading rate of 50 mg/L. Thereafter slow stirring was applied for 48 hours in a closed vessel to

reach a maximum concentration of dissolved test item in the test water. The mixing 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. After this treatment, stirring was

stopped for 24 hours as a precaution to allow phase separation in case test item droplets had

been mixed into the water column during stirring. Following cessation of mixing and the period

of settling, the lower aqueous and equilibrated phase was carefully separated from the nondissolved

upper test item phase through the tap at the bottom of the vessel. This equilibrated

aqueous phase with a loading rate of 50 mg/L, containing dissolved test item only, was used as

highest test concentration and considered to represent the aqueous saturation concentration in

test media of the test item. The highest test concentration was subsequently diluted with test

water to obtain the following test media: the dilutions 1:5.1, 1:3.4, 1:2.3 and 1:1.5. Additionally,

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

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.

The measured concentrations of the test item GR-86-6599 in the test media of all test

concentrations at the start and end of the two test medium renewal periods are shown in the

table below. The results demonstrate the correct preparation of the test media, i.e. the spacing

factor of 1.5 between the different test concentrations was sufficiently met. At the end of the

test medium renewal periods, 65 to 103% of the initially measured values were found.

From these results the mean measured concentrations were calculated as follows: for each test

medium renewal period (i.e. Day 0 to Day 1, Day 1 to Day 2), the mean concentration was

calculated as the geometric mean of the test item concentrations measured at the start and the

end of the test medium renewal period. From the two geometric mean values obtained, the mean

measured test item concentration during the test period of 48 hours was calculated as an

arithmetic mean.

The biological results are based on the mean measured concentrations.

After 48 hours of exposure, no immobilized test organisms were determined in the control and

up to and including the highest test concentration of 123 μg/L, corresponding to the saturation

concentration of the test item.

As no toxic effect was observed, the 48-hour EC50 and 48-hour EC100 were determined directly

from the raw data to be >123 μg/L and the EC0 and NOEC to be ≥123 μg/L.

In conclusion the test item GR-86-6599 had no acute toxic effects on Daphnia magna in a

48-hour semi-static test up to the highest test concentration of 123 μg/L, corresponding to the

saturation concentration of the test item.

Description of key information

The acute toxicity of the test item GR-86-6599 to Daphnia magna was determined in a 48-hour semi-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.


 


As no toxic effect was observed, the 48-hour EC50 and 48-hour EC100 were determined directly from the raw data to be >123 μg/L and the EC0 and NOEC to be ≥123 μg/L.


In conclusion the test item GR-86-6599 had no acute toxic effects (immobility and observed appearance and behaviour) on Daphnia magna in a 48-hour semi-static test up to the highest test concentration of 123 μg/L, corresponding to the saturation concentration of the test item.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
EC50
Effect concentration:
> 123 µg/L

Additional information

According to the EU CLP regulation (No 1272/2008 and its adaption 286/2011), Scentaurus Clean doesn't need to be classified as Hazardous to the Aquatic Environment Acute 1 nor Chronic classifications (Acute results represent saturation concentration of the test item).