Reaction products of diphenylamine with nonene, branched

Sediment toxicity

Link to relevant study record(s)

Reference

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 225 (Sediment-Water Lumbriculus Toxicity Test Using Spiked Sediment)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations in sediment: 1000, 316, 31.6, 10, 3.16 mg/kg dw
- Sampling interval: day 0, 7, 28
- Sample storage before analysis: at room temperature, prepared samples were stored in an autosampler at room temperature until analysis
- analysed compartments: aqueous layer, pore water, sediment

Vehicle:

yes

Remarks:

Acetonitrile

Details on sediment and application:

PREPARATION OF SPIKED SEDIMENT
The respective test item amount was weighed for each test item concentration and dissolved in Acetone. The appropriate amount of spiking solution (2.5 mL per replicate) was thoroughly mixed with a subset of the artificial sediment (10 g per replicate). After complete evaporation of the solvent, the spiked portion of artificial sediment was thoroughly mixed with the remaining sediment. Demnineralised water was added to the artificial sediment to adjust the humidity of the artificial sediment to a moisture of 30 %.
Subsequently, the test medium was thoroughly mixed to ensure a homogenous distribution and about 45 g soil dry weight was filled into test vessels.

Artificial sediment containing:
- 4.2 % peat, air-dried and finely ground
- 20 % kaolin, with a kaolinite content > 30
- 75 % quartz sand (> 50 % particles sized 50-200 pm)
- 0.45 % calcium carbonate (CaCO3) to achieve a pH of 6.62
The organic carbon content of the final mixture was 2.23 %.
45 g dry weight per vessel / ca. 1.0 - 3.0 cm from the ground.
Overlying water: Same as for holding. Volume per replicate: 150 mL. Prior to the start of the exposure, the pH-value, the oxygen content and hardness of the water medium were determined.
Equilibration period: 2 days

Test organisms (species):

Lumbriculus variegatus

Details on test organisms:

Test system: Lumbriculus variegatus (Müller), Lumbriculidae, Oligochaeta
Reason for the selection of the test system: Lumbriculus variegatus is the recommended species according to the guideline.
Origin: Bundesanstalt fOr Gewässerkunde (BfG), Am Mainzer Tor 1, 56068 Koblenz, Germany
Breeding: Breeding is performed at the test facility at 20 ± 2 00 and diffuse light (100 - 500 lx, 16 h photoperiod daily). The dissolved oxygen concentration is > 70 % of the air saturation
value corresponding to 6.2 Mg 02/L. All worms used in the test originated from the same delivery of the supplier and the same stock. They were not used in another test.
Medium: Breeding of L. variegatus is performed on quartz sand in reconstituted water containing 125 mL from each stock solution poured in a can and filled up to 25 L with demineralised water.
Feeding: TetraMin8 flake food (TETRA) is provided twice per week.

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Hardness:

Day 0: 386 - 512 mg/L CaCO3
Day 28: 257 - 327 mg/L CaCO3

Test temperature:

19.7 - 22.2 °C
(Day 0: 19.7 - 20.2 °C; Day 7: 21.9 - 22.2 °C; Day 14: 20.3 - 21.1 °C; Day 21: 20.1 - 20.5 °C; Day 28: 20.7 - 21.1 °C)

pH:

Sediment: 6.62 (at test start)
Water: 7.48 - 8.36 (troughout the test; Day 0: 7.60 - 8.36; Day 7: 7.48 - 7.88; Day 14: 7.55 - 7.82; Day 21: 8.09 - 8.26; Day 28: 7.61 - 7.88)

Dissolved oxygen:

6.29 - 8.76 mg O2/L (Day 0: 7.35 - 8.76; Day 7: 6.29 - 8.52; Day 14: 8.30 - 8.73; Day 21: 8.19 - 8.72; Day 28: 7.96 - 8.73; reported in mg O2/L)

Ammonia:

0.07 (start) - 0.58 mg/L

Nominal and measured concentrations:

The study was performed by spiking artificial sediment with the test item concentrations 3.16 - 10.0 - 31.6 - 100 -316 - 1000 mg/kg sediment dry weight.
The measured concentrations of the test item in the sediment were in the range from 84% to 105% at day 0, from 90% to 123% at day 7 and from < LOQ to 97% of the nominal concentrations at day 28. Minor recoveries of the concentrations of the test item in the aqueous phase and pore water could be determined. Control and solvent control samples were < LOQ.

Details on test conditions:

EFFECT PARAMETERS MEASURED
Visual observations of behavioural differences to the control were recorded three times per week. The endpoints of the study as determined after day 28 were the number of surviving worms per replicate and the total dry biomass of worms per replicate. Worms were considered dead if showing no response to a mechanical stimulus, signs of decomposition or by absence. Living worms were assigned as complete worms or incomplete worms.

PHYSICAL CHEMICAL PARAMETERS
Water quality analysis: Temperature, pH-value and oxygen content of the overlying water were measured weekly as well as at the test start and test end in one additional replicate of the controls and each test item concentration. The temperature between vessels did not deviate by more than 1 °C at one point of time. Aeration of the test vessels was checked at least each working day. Total hardness of the overlying water was measured in one additional replicate of the controls and of all test item concentrations at the start and the end of the test. Ammonium (as NH4+-N) of the overlying water was measured three times per week as well as at test start and test end in one additional replicate of the controls and each test item concentration. -
Environmental conditions: During the test, the room temperature was recorded continuously with a thermohygrograph. Light intensity was determined prior to insertion of test organisms.

CHRONOLOGICAL TEST DESCRIPTION
Preparation of the sediment: Artificial sediment was produced and adjusted to the recommended pH range and to the recommended total organic carbon content. Determination of the sediment moisture
Day -13: Synchronisation of the worms-
Day -2 (experimental starting): Application as described above; Test vessels were filled with the sediment; Determination of the pH, hardness and oxygen content of the overlying water; Reconstituted water was added as described above; Aeration of the test vessels-
Day 0: Determination of pH, temperature and oxygen content of the overlying water Analysis of hardness and ammonium in the overlying water Insertion of synchronised worms into the test vessels Determination of mean dry weight of the worms-
Three times per week: Visual observation of behavioural effects on the worms Analysis of the ammonium content in the overlying water-
Once per week: Determination of pH, temperature and oxygen content in the overlying water-
Day 0, 7, 28: Analytical determinations of the controls and all test item concentrations from additional replicates
Day 28: Determination of pH, temperature and oxygen content in the overlying water; Analysis of hardness and ammonium in the overlying water; Visual observation of behavioural effects; Determination of the total worm number of each test vessel; Determination of the worm dry mass per test vessel.

Reference substance (positive control):

yes

Remarks:

Potassium chloride (2018-03-26/2018-04-26)

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

858 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: Confidence Intervall 628 - >1000 mg/kg DW

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

678 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Confidence Intervall 401 - > 1000 mg/kg DW

Details on results:

Mortality I Reproduction of Worms:
No mortality of worms was observed in the controls and all test item concentrations after exposure to the test item over 28 days. Since no statistically significant difference was found between the control and the solvent control, both controls were pooled for further evaluation. The total number of worms and thus the reproduction did not significantly differ compared to the pooled control in the test item concentrations 3.16 to 100 mg/kg sediment dry weight. However, at the test item concentrations 316 to 1000 mg/kg sediment dry weight the total number of worms and thus the reproduction was significantly reduced.

Behaviour and Pathological Symptoms:
No evident changes in the worm behaviour or pathological symptoms were observed in the control as well as in all test item concentrations during the 28 days ofexposure.

Dry Biomass of the Worms per Replicate:
At test start the worms had a body weight of 10.9 mg dry weight/10 worms (mean weight of two determinations). At test end, the mean biomass dry weight of the worms was not statistically significantly different at the test item concentrations 3.16 to 100 mg/kg soil dry weight compared to the pooled control. However, at the test item concentration 316 and 1000 mg/kg soil dry weight the mean biomass dry weight of the worms was statistically significantly reduced.

Results with reference substance (positive control):

A reference item study is performed once a year with Potassium chloride. The most recent reference item test was carried out from 201 8-03-26 and 2018-04-2. The study was conducted with four reference item concentrations of 1.0 -2.0 -4.0 - 8.0 g/kg sediment dry weight.
The effecrs are summarised as follows:
- NOEC: total worm number: 2 g/kg d.w.
- NOEC: biomass: 1 g/kg d.w.
- LOEC: total worm number: 4 g/kg d.w.
- LOEC: biomass: 2 g/kg d.w.
- EC50: total worm number: 3.04 g/kg d.w.
- EC50: biomass: 2.63 g/kg d.w.

Reported statistics and error estimates:

NOEC/LOEC-values and statistical analyses :
Since no statistically significant difference was determined between the control and the solvent control, both controls were
pooled for further evaluation:
Biomass:
- Shapiro-Wilk's Test on Normal Distribution
- Levene's Test on Variance Homogeneity (with Residuals)
- Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm
Reproduction:
- Shapiro-Wilk's Test on Normal Distribution
- Levene's Test on Variance Homogeneity (with Residuals)
- Trend Analysis by Contrasts (Monotonicity of Concentration/Response
- Williams Multiple Sequential t-test Procedure

The a-value (acceptable probability of incorrectly concluding that there is a difference) is a = 0.05.

EC-values and statistical analyses:
The EC10/20/50 - values of biomass and reproduction were determined running Sigmoidal dose-response.

Validity criteria fulfilled:

yes

Conclusions:

After 28 days of exposure, the test item induced no mortality in all test item concentrations. Since no statistically significant difference between the control and the solvent control was determined, both controls were pooled for further evaluation. The total number of worms and thus the reproduction did not statistically significantly differ compared to the pooled control in the test item concentrations 3.16 to 100 mg/kg sediment dry weight. However, at the test item concentrations 316 and 1000 mg/kg sediment dry weight the total number of worms was statistically significantly reduced.
Compared to the pooled control, the total biomass per replicate was not statistically significantly different in the test item concentrations 3.16 to 100 mg/kg sediment dry weight. However, at the test item concentrations 316 and 1000 mg/kg sediment dry weight the total biomass per replicate was statistically significantly reduced.
A NOEC reproduction = 100 mg/kg DW as well as a NOEC biomass = 100 mg/kg DW were determined.
All validity criteria were fulfilled.

Executive summary:

NOEC reproduction = 100 mg/kg DW

NOEC biomass = 100 mg/kg DW

Description of key information

Toxic effects have been observed in studies with sediment dwelling organisms.

NOEC biomass = 100 mg/kg DW

NOEC reproduction = 100 mg/kg DW

Additional information

After 28 days of exposure, the test item  induced no mortality in all test item concentrations. Since no statistically significant difference between the control and the solvent control was determined, both controls were pooled for further evaluation. The total number of worms and thus the reproduction did not statistically significantly differ compared to the pooled control in the test item concentrations 3.16 to 100 mg/kg sediment dry weight. However, at the test item concentrations 316 and 1000 mg/kg sediment dry weight the total number of worms was statistically significantly reduced.

Compared to the pooled control, the total biomass per replicate was not statistically significantly different in the test item concentrations 3.16 to 100 mg/kg sediment dry weight. However, at the test item concentrations 316 and 1000 mg/kg sediment dry weight the total biomass per replicate was statistically significantly reduced.

A NOEC reproduction = 100 mg/kg DW as well as a NOEC biomass = 100 mg/kg DW were determined.