N,N-Bis-(4-biphenyl)amine

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:

Sep 18 - Nov 04, 2019

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)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The samples were taken from the biological phase of the study. Collecting, storage and handing over of the samples were the Study Director’s responsibility.
Duplicate samples from the freshly prepared test media of the only test concentration and the control were taken at the start of the test and at day 1.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the aged test media of the only test concentration and the control were collected at day 1 (after 24 hours of exposure) and at the end of the test by pouring together the contents of the test beakers of each treatment.
All samples were diluted by a factor of 2 with acetonitrile.
Additional samples of the control and the dilution solvent were taken at each sampling test start and test end without any sample treatment.
All samples were stored in a freezer (≤ - 20 °C), protected from light, until analysis was performed.

Vehicle:

no

Details on test solutions:

Reconstituted Water (Elendt "M4"): Analytical grade salts and additives were added at the following nominal concentrations in deionised water (conductivity < 5 μScm-1):
-------------------------------------------------------------------------------------------------------------------------
Main Compounds Trace Elements Vitamins
-------------------------------------------------------------------------------------------------------------------------
CaCl2·2 H2O 293.80 mg/L H3BO3 2860 μg/L Thiamin HCl 75.0 μg/L
MgSO4·7 H2O 123.30 mg/L MnCl2·4 H2O 361 μg/L Cyanocobalamin (B12) 1.0 μg/L
KCl 5.80 mg/L LiCl 306 μg/L Biotin (B6) 0.75 μg/L
NaHCO3 64.80 mg/L RbCl 71 μg/L
Na2SiO3·9 H2O 10.00 mg/L SrCl2·6 H2O 152 μg/L
NaNO3 0.27 mg/L NaBr 16 μg/L
KH2PO4 0.14 mg/L Na2MoO4·2 H2O 63 μg/L
K2HPO4 0.18 mg/L CuCl2·2 H2O 17 μg/L
ZnCl2 13 μg/L
CoCl2·6 H2O 10 μg/L
KI 3.3 μg/L
Na2SeO3 2.2 μg/L
NH4VO3 0.6 μg/L
Na2EDTA·2 H2O 2.5 mg/L
FeSO4·7 H2O 1.0 mg/L
-------------------------------------------------------------------------------------------------------------------------
Water Hardness: 2.5 mmol/L (= 250 mg/L) as CaCO3 Alkalinity: 0.9 mmol/L

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Daphnia magna, Clone 5
- Justification for species other than prescribed by test guideline: accepted by Guideline
- Source: in house
- Age of parental stock (mean and range, SD): 1.75 - 18.25 hours
- Feeding during test: no
- Food type: -
- Amount: -
- Frequency: -

ACCLIMATION
- Acclimation period: no
- Acclimation conditions (same as test or not): -
- Type and amount of food: -
- Feeding frequency: -
- Health during acclimation (any mortality observed): -

QUARANTINE (wild caught)
- Duration: -
- Health/mortality: -

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

250 mg/L

Test temperature:

20.0 - 20.5 °C

pH:

7.7 - 7.9

Dissolved oxygen:

6.5 to 8.6 mg/L

Salinity:

-

Conductivity:

-

Nominal and measured concentrations:

nominal: 100 mg/L
analytical: below LOQ of 0.005 mg/L

Details on test conditions:

The test item was not well soluble in test water. To avoid physical effects of undissolved test item on the daphnids, no concentration above the solubility limit of the test item in test water was tested. Therefore, a supersaturated stock solution of nominal 100 mg test item/L was prepared by suspending 102.7 and 100.5 mg of test item in 1027 and 1005 mL test water, respectively. The stock solutions were stirred for 24.3 and 23.8 hours in the dark, respectively to dissolve as much test item as possible. Then, the undissolved test item was separated by filtration (0.2 μm cellulose acetate filter) and the filtrate was used as test medium.
The test media was prepared just before introduction of the daphnids (= start of the test) and test medium renewal on Day 1. The test vessels were saturated one hour before use.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Values refer to nominal test concentrations of a filtrate of 100 mg test item/L. The concentration analytically measured was below the limit of quantification of the analytical method (LOQ of 0.005 mg test item/L).

Results with reference substance (positive control):

Reference Substance: Potassium Dichromate
EC50 (48h) = 0.542 mg/L

Biological Results

After 48 hours of exposure no immobilisation of the test animals was observed in the control and the only test item concentration of the filtrate of 100 mg test item/L.

Nominal Concentration / [mg test item / L]	% of immobilised daphnids after
	24 hours	48 hours
Control	0	0
100	0	0

	Effect Concentrations / [mg/L] after
	24 hours	48 hours
EC50	> 100	> 100
95 % CI	n.d.	n.d.
EC20	> 100	> 100
95 % CI	n.d.	n.d.
EC10	> 100	> 100
95 % CI	n.d.	n.d.
NOEC	≥ 100	≥ 100
LOEC	> 100	> 100

CI: Confidence interval n.d.: not determinable NOEC and LOEC were determined directly from the raw data.

Validity criteria fulfilled:

yes

Conclusions:

The toxic effect of the test item to Daphnia magna was assessed in a semi-static limit test. The 48-hour NOEC was determined to be at least the filtrate of nominal 100 mg test item/L. The NOEC might even be higher than this concentration, but concentrations in excess of the limit of solubility have not been tested according to the recommendations of the test guidelines. The 48-hour LOEC and the 48-hour EC50 were clearly higher than the filtrate of nominal 100 mg test item/L. These values could not be quantified due to the absence of toxicity of the test item up to the filtrate of nominal 100 mg test item/L, which is the concentration representing the limit of solubility. The initial concentrations and the maintenance of the exposure concentrations during the test were examined in the analytical part. No test item concentrations above the limit of quantification of the analytical method were found. All reported results refer to the nominal concentrations.

Executive summary:

The purpose of this study was to determine the influence of the test item on the mobility of Daphnia magna.
For this purpose, young daphnids (< 24 hours old) were exposed in a semi-static test for 48 hours to the only concentration of nominal 100 mg/L under defined conditions. The recorded effects were the mobility of the daphnids after 24 and 48 hours.
This limit-test was performed in compliance with the test guidelines in order to demonstrate that the test item has no toxic effects on the daphnids up to this concentration.
The test method of application and the test system are recommended by the test guidelines and Daphnia magna is the recommended test species.
The purpose of the analytical part of this study was to verify the concentration of the test item in the test medium.

The informtion for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 202.
This study encompassed two treatment groups (one test item concentration at nominal 100 mg/L and one control) each containing 20 individuals. The mobility of the daphnids was determined in a semi-static 48-hour test by visual observation after 24 and 48 hours. Due to the low water solubility limit of the test item a filtrate of nominal 100 mg/L was tested. The measured concentration was below the LOQ of 0.005 mg test item/L. No concentrations above the solubility limit of the test item in the used test water were tested. Additionally, a control was tested in parallel. Thus, a limit test in accordance with OECD Guideline 202 was performed to demonstrate that the test item has no toxic effect on daphnids up to the solubility limit in the used test water.
The exposure was analytically verified using an HPLC method with UV detection.

Biological Results

After 48 hours of exposure no immobilisation of the test animals was observed in the control and the only test item concentration of the filtrate of 100 mg test item/L.
The 48 -h EC50 was determined to be > 100 mg/L and the 48 -h NOEC was determined to be >= 100 mg/L. The values refer to nominal test concentrations of a filtrate of 100 mg test item/L. The concentration analytically measured was below the limit of quantification of the analytical method (LOQ of 0.005 mg test item/L).

Analytical Results

The quantification of the test item in the test samples was performed using liquid chromatography with UV detection. In the test media of the only test concentration (filtrate of test item stock solution of nominal 100 mg test item/L) the measured values are below the limit of quantification of the analytical method (LOQ = 0.005 mg test item/L).

Conclusion

The toxic effect of the test item to Daphnia magna was assessed in a semi-static limit test. The 48-hour NOEC was determined to be at least the filtrate of nominal 100 mg test item/L. The NOEC might even be higher than this concentration, but concentrations in excess of the limit of solubility have not been tested according to the recommendations of the test guidelines. The 48-hour LOEC and the 48-hour EC50 were clearly higher than the filtrate of nominal 100 mg test item/L. These values could not be quantified due to the absence of toxicity of the test item up to the filtrate of nominal 100 mg test item/L, which is the concentration representing the limit of solubility. The initial concentrations and the maintenance of the exposure concentrations during the test were examined in the analytical part. No test item concentrations above the limit of quantification of the analytical method were found. All reported results refer to the nominal concentrations.

Description of key information

OECD 202:

No adverse effects to daphnia magna were observed up to the limit of water solubility.

EC50 (48 h) > 100 mg/l (nominal concentration)

Additional information

The toxic effect of the test item to Daphnia magna was assessed in a semi-static limit test. The 48-hour NOEC was determined to be at least the filtrate of nominal 100 mg test item/L. The NOEC might even be higher than this concentration, but concentrations in excess of the limit of solubility have not been tested according to the recommendations of the test guidelines. The 48-hour LOEC and the 48-hour EC50 were clearly higher than the filtrate of nominal 100 mg test item/L. These values could not be quantified due to the absence of toxicity of the test item up to the filtrate of nominal 100 mg test item/L, which is the concentration representing the limit of solubility. The initial concentrations and the maintenance of the exposure concentrations during the test were examined in the analytical part. No test item concentrations above the limit of quantification of the analytical method were found. All reported results refer to the nominal concentrations.