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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:
2017-10-17 to 2017-10-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well documented guideline study with GLP and no deviations.
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
OECD-Guideline 202 for Testing of Chemicals (2004)
Deviations:
no
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media were performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose the organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture (e.g. UVCB) and is poorly soluble in water and in the permitted solvents.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Test item: Tall oil pitch, reaction product with triethylene glycol
CAS number: 2135769-54-7
Batch number: LABO 16-02
Purity: 100% (UVCB substance)
Water solubility: Poorly soluble
Appearance: Viscous (liquid), black
Stability under test conditions: Not specified
Expiry date: 2018-11-14
Analytical monitoring:
yes
Details on sampling:
At the start of the exposure and at renewal (0 and 24 hours), samples of the fresh media were taken after preparation of the WAF and analyzed. At renewal and at the end of the exposure (24 and 48 hours), samples of the 24-hours old media were taken from additional replicates with test media, but without daphnids and analyzed.
Vehicle:
no
Details on test solutions:
Limit loading level
A WAF with a loading level of 100 mg/L was tested in a limit test. The WAF is based on the results of a non-GLP preliminary range finding test conducted at the test facility under semi-static conditions.

Water Accommodated Fraction (WAF)
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media were performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere, is to expose the organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture (e.g. UVCB) and is poorly soluble in water and in the permitted solvents. Using this approach, the test solution will be prepared by mixing the test item with dilution water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. At the completion of mixing and following a settlement period, the test item phase was separated by siphoning and the test organisms were exposed to the equilibrated aqueous phase, i.e. the WAF. Exposure was expressed in terms of the initial nominal concentration of the test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of the test item in the WAF.

Per definition of the WAF, all terms related to concentration level have to be given as loading level because partly dissolved compounds and mixtures cannot be related to concentrations.

Preparation of the water accommodated fraction
The WAF was prepared one day prior to the start of the exposure (at -24 hours) and the renewal of the test solutions (at 0 hours) in a glass flask. An appropriate amount of the test item was weighed out on a glass slide and inserted into a brown glass flask filled with an appropriate amount of dilution water. This dispersion was shaken for 24 hours at 20 rpm at room temperature. After completion of shaking and following a separation phase of at least 1 hour of standing, the aqueous phase or WAF was removed from the approximate center of the water body by siphoning.

Control
Dilution water without test item incubated under the same conditions as the test groups
Test organisms (species):
Daphnia magna
Details on test organisms:
Test system: Daphnia magna STRAUS (Clone 5)

Reason for the selection of the test system: Daphnia magna is the preferred species in accordance with the test guideline and is bred at the test facility.

Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany

Breeder: Noack Laboratorien GmbH, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany

Culture: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 +/- 2 °C, in an incubator, 16 hours illumination, light intensity of max. 1500 lx

Culture medium: Elendt M4 is used. The composition is modified to a total hardness of approximately 160 to 180 mg CaCO3/L.

Culture feeding: The culture daphnids are fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 106 cells/mL. The algae are cultured at the test facility.

Origin of the food algae: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
175-0180 mg CaCO3/l
Test temperature:
19 °C
pH:
7.39 - 7.90
Dissolved oxygen:
7.58 - 8.85 mg/l
Conductivity:
440-442 µS/cm
Details on test conditions:
Test method The study was performed under semi-static conditions (with a renewal of the test solutions after 24 hours) as a worst case exposure, because the test item is an UVCB substance and the unspecific analytical method, which was used for the analytical monitoring, cannot confirm the chemical stability of the test item.

Test duration: 48 hours
Test vessels: Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses
Test volume: 20 mL
Dilution water: Same as culture medium
Number of daphnids: 20 daphnids, divided into 4 replicates, each with 5 daphnids were used for the WAF and the control.
Age of the daphnids at the start of the exposure: Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.
Acclimatization: Acclimatization was not necessary, because the dilution water was equivalent to the culture medium.
Application: 20 g test solution per replicate were weighed out into each test vessel. This corresponds to 20 mL per test vessel. The daphnids were transferred using a plastic pasteur pipette with a minimum amount of dilution water (start of the exposure) or test solution (renewal of the test solutions).
Renewal of the test solutions: The test solutions were renewed after 24 hours. For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette (see ‘Application’).
Test temperature (target): 18 - 22°C, constant within ± 1°C
Illumination (target): Diffuse light, light intensity of max. 1500 lx
Photoperiod (target): 16/8 hours light/dark cycle
Feeding: The daphnids were not fed during the study.

Reference test
A reference test was conducted as an acute immobilization test in Elendt M4 medium under static conditions with a test duration of 24 hours once per month in order to prove the validity of the test system and test conditions at the test facility. The results of the most recent test are presented in section 0.

Reference item: Potassium dichromate p.a. (SIGMA-ALDRICH)
Purity: 99.0%
Batch number: MKBV0900V
Expiry date: 2021-11-25
Test concentrations: 0.500 – 1.00 – 2.00 – 4.00 mg/L
Ranges of validity: EC50 (24 hours): 0.6 - 2.4 mg/L, according to AQS P 9/2 (clone 5),
EC50 (24 hours): 0.6 - 2.1 mg/L, according to OECD 202 (clone A)
Exposure phase: 2017-10-05 to 2017-10-06
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Non-GLP Preliminary Range Finding Test
A non-GLP preliminary range finding test under semi-static conditions over a period of 48 hours was conducted at the test facility with three water accommodated fractions (WAF) with a nominal loadings of 1, 10 and 100 mg test item/L. The loading levels 1 and 10 mg/L of the test item were visually clear throughout the exposure period and had a Tyndall-effect, whereas turbidity was observed in the WAF 100 mg/L. The preliminary range finding test was conducted under diffuse light conditions (light intensity of max. 1500 lx, 16/8 hours light:dark cycle). In the range finding test, two replicates per treatment group and control, each with ten daphnids, were tested.

Definitive Test
The effect levels (Immobilisation, EL10 / 50 / 100) were based on the nominal loading levels of the test item. Per definition of the WAF, all terms related to concentration levels have to be given as loading levels because partly dissolved compounds and mixtures cannot be related to concentrations. The WAF was turbid throughout the exposure period.

The limit loading level of the test item Tall oil pitch, reaction product with triethylene glycol and the control were analytically verified via analysis of total organic carbon (TOC, according to DIN EN 1484) at the start of the exposure intervals (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours). At the end of the exposure intervals (24 and 48 hours), samples of the old media were taken for analysis from additional replicates, which were prepared with test media, but without daphnids. The measured carbon concentration in the WAF in the fresh media at the start of exposure and the renewal after 24 hours were 14.3 and 3.57 mg/L. The measured carbon concentration in the WAF in the old media at the renewal after 24 hours and at the end of the exposure were 11.0 and 2.87 mg/L. The limit of quantification of the analytical method is set to 2.00 mg/L.

The measured water quality parameters (i.e. pH-value, dissolved oxygen concentration, total water hardness and water temperature) were within the acceptable limits.

The study was performed according to OECD Guideline 202 (2004). The validity criteria were fulfilled:
In the control group, no daphnids were immobilized or showed any signs of disease or stress, e.g. discoloration or unusual behavior such as trapping on the surface of the water, during the 48-hour test period (required: not more than 10% of the daphnids in the control).

The dissolved O2 concentration in the old media at the end of the exposure was ≥ 7.58 mg/L (required: ≥ 3 mg/L at the end of the exposure) in the test vessels of all tested WAFs and the control.

In the WAF with a nominal loading of 100 mg/L of the test item Tall oil pitch, reaction product with triethylene glycol, no effects on Daphnia magna were observed.

Table 2. Immobilization Rates in the non-GLP Preliminary Range Finding Test (n = 20, divided into 2 replicates with 10 daphnids each)

Nominal loading level of the test item in the WAF [mg/L]

IMMOBILIZATION [%]

24 hours

48 hours

Replicates

Replicates

1

2

Mean

1

2

Mean

100

10

0

5

10

0

5

10

0

0

0

0

0

0

1

0

0

0

0

0

0

Control

0

0

0

0

0

0

Table 3. Measured TOC concentrations during the non-GLP Range finding Test.

Sampling date

2017-05-16 0 hours Start of the exposure interval

2017-05-17 24 hours End of the exposure interval

2017-05-17 24 hours Start of the exposure interval

2017-05-18 48 hours End of the exposure interval

Nominal loading level of the test item in the water accommodated fraction [mg/L]

Total Organic Carbon (TOC)

Meas. conc. [mg C/L]

Meas. conc. [mg C/L]

Meas. conc. [mg C/L]

Meas. conc. [mg C/L]

100

13.6

13.0

7.92

6.03

10

4.35

4.60

3.95

4.28

1

2.35

2.49

3.19

3.80

Control

2.08

3.00

2.83

< 2.00

Meas. conc.=measured TOC concentration, mean value of 2 injections

Table 4. Immobilization Rates after 24 and 48 hours of Exposure during the Definitive Test (n = 20, divided into 4 replicates with 5 daphnids each)

Nominal loading level
of the test item in the water accommodated fraction
[mg/L]

IMMOBILIZATION [%]

24 hours

48 hours

Replicates

Replicates

1

2

3

4

Mean

1

2

3

4

Mean

100

0

0

0

0

0

0

0

0

0

0

Control

0

0

0

0

0

0

0

0

0

0

Table 5.      Absolute Numbers of immobile Daphnids after 24 and 48 h of Exposure in the Definitive Test (n = 20, divided into 4 replicates with 5 daphnids each)

Nominal loading level
of the test item in the water accommodated fraction
[mg/L]

Number of immobile Daphnids / Total number of Daphnids

24 h

48 h

Replicates

Replicates

1

2

3

4

MV

1

2

3

4

MV

100

0 / 5

0 / 5

0 / 5

0 / 5

 0 / 20

0 / 5

0 / 5

0 / 5

0 / 5

 0 / 20

Control

0 / 5

0 / 5

0 / 5

0 / 5

 0 / 20

0 / 5

0 / 5

0 / 5

0 / 5

0 / 20

Table 6. Measure TOC concentrations during the Definitive Test

Sampling date

2017-10-18

Fresh media, 0 hours

2017-10-19

Old media, 24 hours

2017-10-19

Fresh media, 24 hours

2017-10-20

Old media, 48 hours

Nominal test item loading [mg/L]

Total Organic Carbon (TOC)

Measured concentration1) [mg C/L]

Measured concentration1) [mg C/L]

Measured concentration1) [mg C/L]

Measured concentration1) [mg C/L]

100

14.3

11.0

3.57

2.87

Control

<LOQ* (0.823)

<LOQ* (0.733)

<LOQ* (1.03)

<LOQ* (0.778)

Validity criteria fulfilled:
yes
Conclusions:
In the WAF with a nominal loading of 100 mg/L of the test item Tall oil pitch, reaction product with triethylene glycol, no effects onDaphnia magnawere observed. Therefore, the Therefore, the EL10 / 50 / 100-values were all >100 mg/l.
Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item Tall oil pitch, reaction product with triethylene glycol (batch number:LABO16-02) were determined at the test facility according to OECD 202 (2004). The study was conducted under semi-static conditions over a period of 48 hours with one Water Accommodated Fractions (WAF) with a nominal loading of 100 mg/L. In the WAF with a nominal loading of 100 mg/L of the test item Tall oil pitch, reaction product with triethylene glycol, no effects on Daphnia magna were observed. Therefore, the Therefore, the EL10 / 50 / 100-values were all >100 mg/l.

Description of key information

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item Tall oil pitch, reaction product with triethylene glycol (batch number:LABO16-02) were determined at the test facility according to OECD 202 (2004). The study was conducted under semi-static conditions over a period of 48 hours with one Water Accommodated Fractions (WAF) with a nominal loading of 100 mg/L. In the WAF with a nominal loading of 100 mg/L of the test item Tall oil pitch, reaction product with triethylene glycol, no effects on Daphnia magna were observed. Therefore, the Therefore, the EL10 / 50 / 100-values were all >100 mg/l.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
100 mg/L

Additional information