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Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2019-01-07 to 2019-01-10, with the definitive exposure phase from 2019-01-08 to 2019-01-10

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

GLP compliance:

yes

Analytical monitoring:

no

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. Per definition of the WAF, all terms related to the concentration level have to be given as loading level because partly dissolved compounds and mixtures cannot be related to concentrations. The test item loading was chosen on the results of a non-GLP preliminary range finding test conducted at the test facility under semi-static conditions.

Water Accommodated Fraction
A water accommodated fraction (WAF) was prepared because the test item is a UVCB substance with compounds of different water solubility. This procedure is in accordance with the OECD guidance document No. 23 (2018). Using this approach, aqueous media were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase.

Preparation of the WAF
One WAF was prepared with a nominal loading of 100 mg/L. An appropriate amount of the test item was weighed out and transferred into a glass flask with an appropriate amount of demineralized water. The test item dispersion was shaken for 24 hours with 20 rpm at room temperature. After completion of shaking and a separation phase of at least 1 hour of standing at room temperature, the WAF was removed by siphoning from the approximate center of the water body. After siphoning, the WAF was checked via laser beam (Tyndall effect) for undissolved test item. The WAF had a Tyndall effect, but was visually clear and colorless throughout the exposure period. The following WAF was used in the test after addition of the components of the dilution water.
The preparation of the WAF was carried out beginning one day prior to the start of the exposure (day -1) and one day prior to the renewal of the test solutions (day 0).

Control
Dilution water without test item incubated under the same conditions as the test group.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system: Daphnia magna STRAUS, obtained from continuous laboratory cultures.

Reason for the selection of the test system
Daphnia magna is the preferred species in accordance with the test guideline.

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, according to OECD 202, Annex 3 (2004), is used. The composition of the culture medium is presented in the table below.

 

Composition of the Culture Medium Elendt M4
according to OECD 202, Annex 3 (2004)

Component Concentration [mg/L]
CaCl2 x 2 H2O 294
MgSO4 x 7 H2O 123
KCl 5.80
NaHCO3 64.8
Na2SiO3 x 5 H2O 7.47
NaNO3 0.274
KH2PO4 0.143
K2HPO4 0.184
Na2EDTA x 2 H2O 2.50
FeSO4 x 7 H2O 0.996
H3BO3 2.86
MnCl2 x 4 H2O 0.361
LiCl 0.306
SrCl2 x 6 H2O 0.152
RbCl 0.0710
NaBr 0.0160
Na2MoO4 x 2 H2O 0.0615
CuCl x 2 H2O 0.0168
ZnCl2 0.0130
CoCl2 x 6 H2O 0.0100
KI 0.00325
Na2SeO3 0.00219
NH4VO3 0.000575
Thiaminhydrochloride 0.075
Cyanocobalamin 0.0010
Biotin 0.00075
pH 8.2 to 0.8

Feeding of the culture stocks
The daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus. The algae were cultured at the test facility.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

see pH

Test temperature:

target: 18 - 22°C, constant within ± 1°C
measured: 19 - 20 °C

pH:

Table 6: Water Quality Parameters in the fresh Media at the Start of the Exposure and at the Renewal
(0 and 24 hours)
(measured in one additional replicate (without daphnids) of the WAF and the control)
Nominal loading
of the test item
in the WAF
[mg/L] 0 hours 24 hours
pH-value Dissolved
O2 concentration [mg/L] pH-value Dissolved O2 concentration [mg/L]
100 8.02 8.37 8.22 8.54
Control 7.73 8.44 7.82 9.19


Water Quality Parameters in 24-hours old Media at the Renewal (24 hours) and at the End of the Exposure
(measured in one replicate (containing daphnids) of the WAF and the control)
Nominal loading
of the test item
in the WAF
[mg/L] 24 hours 48 hours
pH-value Dissolved O2 concentration [mg/L] Replicate number pH-value Dissolved O2 concentration [mg/L] Replicate
number
100 7.98 8.31 1 7.16 8.38 1
Control 7.66 7.91 1 7.14 8.42 1


Water Quality Parameters of the Dilution Water at the Start of the Exposure and at the Renewal (0 and 24 hours)
Dilution water
dated: pH-Value Dissolved O2 concentration [mg/L] Temperature [°C] Conductivity [µS/cm] Total hardness [mg CaCO3/L]
0 hours: 2019-01-08 7.73 8.44 19.9 579 291
24 hours: 2019-01-09 7.82 9.19 19.4 612 294

Dissolved oxygen:

see above

Salinity:

Not applicable, freshwater

Conductivity:

Dilution water day 0: 626 [µS/cm]

Nominal and measured concentrations:

WAF with a nominal loading level of 100 mg/L

Details on test conditions:

Test method
The study was performed under semi-static conditions (with a renewal of the test solutions after 24 hours).

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 in each test replicate (see ‘Application’)

Dilution water
Same as culture medium

Number of daphnids and replicates
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 of the daphnids was not necessary, because the dilution water was equivalent to the culture medium.

Application
20 g test solution per replicate was weighed out into each test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water (start of the exposure) or test solution (water renewal) by pipette.

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 substance (positive control):

yes

Duration:

24 h

Dose descriptor:

other: EL10/50/100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

other: EL10/50/100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2018 12-04 to 2018-12-05. For results of the most recent of the monthly performed reference tests, see the table below.

EC50-Value (with Confidence Limits) of the Reference Item Potassium dichromate
based on nominal concentrations [mg/L], (0 - 24 hours)
Current Study EC50 Confidence limits Valid Range
2.03 mg/L 1.00 - 4.00 mg/L 0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A

Reported statistics and error estimates:

The effects of the limit loading level were empirically derived from the observed immobilization rates.
Since no effects on Daphnia magna were observed in the WAF of the test item, no calculations were made. All effect levels (EL10 / 50 / 100) given were based on the nominal loading of the test item. Per definition of the WAF, the exposure has to be expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading level) irrespective of the actual concentration of the test item in the WAF because partly dissolved compounds and mixtures cannot be related to concentrations.

The percentage of immobility, determined in the WAF and in the control after 24 and 48 hours under semi-static conditions.

 

Immobilization Rates [%]after 24 and 48 hours of Exposure in the Definitive Test

(n = 20, divided into 4 replicates with 5 daphnids each)

Nominal loading of the test item in the WAF [mg/L]	IMMOBILIZATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
100	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

 

 

Absolute Numbers of immobileDaphnids after 24 and 48 hours ofExposure in the Definitive Test

                (n = 20, divided into 4 replicates with 5 daphnids each)

Nominal loading of the test item in the WAF [mg/L]	Number of immobile Daphnids / Total number of Daphnids
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	Sum	1	2	3	4	Sum
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

 

Validity criteria fulfilled:

yes

Conclusions:

In the WAF with a limit loading level 100 mg/L of the test item Licocare RBW 300, no effects on Daphnia magna were observed.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of a water accommodated fraction with a limit loading level of 100 mg/L of the test item Licocare RBW 300 were determined at the test facility according to OECD 202 (2004) from 2019-01-07 to 2019-01-10, with the definitive exposure phase from 2019-01-08 to 2019-01-10.

Since the test item is a UVCB substance with compounds of different water solubility and due to the low aqueous solubility of the test item, the test item solution was prepared as a water accommodated fraction (WAF). This procedure is in accordance with the OECD guidance document No. 23 (2018). Per definition, the exposure has to be expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading level) irrespective of the actual concentration of the test item in the WAF because partly dissolved compounds and mixtures cannot be related to concentrations.

One Water Accommodated Fraction (WAF) was prepared with a nominal loading of 100 mg/L and tested in a limit test. The study was performed over a period of 48 hours under semi-static conditions with renewal of test solution after 24 hours as a worst-case exposure scenario. The WAF had a Tyndall effect at the start of the exposure and at renewal, but was visually clear and colorless throughout the exposure period.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to the WAF and the control.

No analytical monitoring of the test item was carried out due to the low solubility of the test item. Therefore,alleffect levels (EL_{10 / 50 / 100}) were given based on the nominal loading of the test item.

The validity criteria of the test guideline were fulfilled.

 

EL_{10 / 50 / 100}-Values

(based on the nominal loading level of the test item)

Effect Level	Test duration [hours]	Licocare RBW 300 Nominal loading level of the test item [mg/L]
EL10 / 50 / 100	24	> 100
	48	> 100

 

Description of key information

In the acute immobilization test withDaphnia magna(STRAUS), the effects of a water accommodated fraction with a limit loading level of 100 mg/L of the test item Licocare RBW 300 were determined at the test facility according to OECD 202 (2004).

In the WAF with a limit loading level 100 mg/L of the test item Licocare RBW 300, no effects on Daphnia magna were observed.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

> 100 mg/L