Tris(2,4-ditert-butylphenyl) phosphite

Sediment toxicity

Link to relevant study record(s)

Reference

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

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)

Version / remarks:

(2007)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The limit concentration level and the control were analytically verified. The concentrations of the test item were analysed on the day of worm insertion (day 0) as well as on day 7 and at test end (day 28) in the sediment layer. Three additional replicates per treatment were set up for the analytical verification on day 0, 7 and 28.

Test organisms (species):

Lumbriculus variegatus

Details on test organisms:

- Test organism: Lumbriculus variegatus (Müller), Lumbriculidae, Oligochaeta.- Origin: Bundesanstalt fUr Gewässerkunde (BfG), Am Mainzer Tor 1, 56068 Koblenz, Germany- Breeding: Breeding is performed at the test facility at 20 ± 2 °C and diffuse light (100 - 500 lx, photoperiod 16 h light daily). The dissolved oxygen concentration is >70 % of the air saturation value corresponding to 6.2 mg O2/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 stock solution (55.8 mg/L CaCl·2H2O; 24.65 mg/L MgSO4·7H2O; 12.95 mg/L NaHCO3; 1.15 mg/L KCl) poured in a can and filled up to 25 L with demineralised water.- Feeding: TetraMin® flake food (TETRA) is provided twice per week.

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Remarks:

(5% peat, air-dried and finely ground; 20% kaolin, with a kaolinite content > 30 %; 75% quartz sand (>50% particles sized 50-200 µm); 0.45% calcium carbonate (CaCO3) to achieve a pH of 6.54)

Limit test:

yes

Duration:

28 d

Exposure phase:

total exposure duration

Hardness:

272 - 322 mg/L (as CaO3)

Test temperature:

20.9 - 22.0 °C

pH:

- Sediment: 6.54 (at the start)- Water: 7.84 - 8.42 (throughout the test)

Dissolved oxygen:

7.98 - 8.59 mg/L O2

Ammonia:

<0.04 (start) - 0.51 mg/L

Nominal and measured concentrations:

- Nominal concentrations: 0 (control) and 1000 mg/kg sediment dry weight (based on range-finding test). A nominal concentration of 1000 mg/kg d.w. corresponds to an amount of 45.0 mg test substance (based on 45 g sediment (DW) and 150 mL dilution water).- Measured concentrations (amount): Day 0:

Details on test conditions:

TEST SYSTEM- Application: Based on the results of the preliminary range finding test, the definitive study was performed as a limit test with a test concentration of 1000 mg/kg sediment dry weight. The limit test item amount was weighed out and mixed thoroughly with a sub-quantity of the artificial sediment. Subsequently, the treated portion of artificial sediment was mixed thoroughly with the remaining sediment. Demineralised water was added to the artificial soil to adjust the humidity of the artificial soil 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.- Control: Overlying water and artificial sediment without test item.- Replicates: Six replicates per control and limit test concentration, one additional replicate each for water quality analyses and one additional replicate for analytical measurements on day 0, 7, 28 each.- Test vessel: 250 mL glass beakers (6 cm diameter)- Test sediment amount/height: 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 test, the pH-value, the oxygen content and hardness of the water medium were determined.- Preparation of test vessels: The test vessels were prepared 2 days prior insertion of the test organisms (see application). Firstly, the glass beakers were filled with the treated sediment. Secondly, the water was added to the beakers without disturbing the sediment. Thirdly, aeration was provided.- Equilibration period: 2 days- Water level: Water levels were topped up with demineralised water each working day during the study to compensate losses due to evaporation.- Aeration: Gentle aeration via Pasteur pipettes. Aeration was checked each working day.- Food Source: Powder of Urtica dioica (stinging nettle, HEINRICH KLENK GMBH & Co KG, 97525 Schwebheim, Germany) was mixed into the sediment at the day of application. The amount was 0.4 % of the sediment dry weight.- Number /Introduction of worms: The worms were inserted in 2 day old test vessels. 10 synchronized worms (similar physiological state) were chosen randomly and inserted per replicate.- Age of the worms: The worms were synchronized 13 days prior to insertion to minimize uncontrolled reproduction and regeneration. Synchronization was achieved by artificial fragmentation of large healthy worms.- Weight of the worms: In order to estimate the mean weight of the worms, two subsamples (10 worms each) were weighed at the start of the test.- Light intensity: 383 ± 72 lux- Photoperiod: 16 h light, 8 h darkEFFECT PARAMETERS MEASUREDVisual observations of behavioural differences to the controls 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 adult (large complete worms) or offspring (small complete worms with regenerated body regions or incomplete worms), if possible.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 replicate of the control and the test item concentration. Aeration of the test vessels was checked each working day. Total hardness of the overlying water was measured in one replicate of the control and of the test item concentration 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 the test start and test end in one replicate of the control and the test item concentration each.- 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. The total organic carbon content and the sediment moisture were determined.- Day -13: Synchronisation of the worms- Day -2: Application as described above (experimental starting); 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 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 (CAS# 7447-40-7)

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: total number of worms (reproduction) and total biomass per replicate

Details on results:

- Mortality / Reproduction of worms: No mortality of worms was observed in the control and the limit test item concentration after exposure to the test item over 28 days. The total number of worms and thus the reproduction did not statistically significantly differ compared to the control.- Behaviour and Pathological Symptoms: No evident changes in the worm behaviour or pathological symptoms were observed in the control as well as in the limit test item concentration during the 28 days of exposure.- Dry Biomass of the Worms per Replicate: At test start the worms had a body weight of 5.9 mg dry weight/10 worms (mean weight of two determinations). At test end, the biomass dry weight of the worms was not statistically significantly different at the limit test item concentration 1000 mg/kg sediment dry weight compared to the control

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 between 2015-03-30 and 2015-04-30. The effects are summarised as follows:- NOEC: total worm number: <1.0 mg/kg d.w. - NOEC: biomass: 1 mg/kg d.w.- LOEC: total worm number: ≤1.0 mg/kg d.w.- LOEC: biomass: 2.0 mg/kg d.w.- EC50: total worm number: 2.6 mg/kg d.w.- EC50: biomass: 2.4 mg/kg d.w.

Reported statistics and error estimates:

- NOEC/LOEC-values and statistical analyses: Williams Multiple Sequential t-Test was used for LOEC and NOEC calculations. When running the test, a Normality Test and an Equal Variance Test were done first. The alpha-value (acceptable probability of incorrectly concluding that there is a difference) is 0.05.

Validity criteria fulfilled:

yes

Description of key information

The long-term toxicity to freshwater sediment organisms was determined in a study according to OECD guideline No 225 and in compliance with GLP criteria (BASF, 2016). In this limit test, sediment worms (Lumbriculus variegatus) were exposed to a nominal test substance concentration of 0 (control) and 1000 mg/kg sediment dry weight for 28 days. Artificial sediment was used which contained 5 % peat, 20 % kaolin and 75 % quartz sand. Test substance concentrations in the sediment layer were analytically verified on the days 0, 7 and 28. No specific analysis in the water phase or pore water was carried out because due to the low water solubility of the test item, this parameter is of minor ecotoxicological relevance. The concentrations of the test item in the sediment samples during the test were 94% at day 0, 68% at day 7 and 16% at day 28 of the nominal concentration. Therefore, all effect values are given as nominal. Visual observations of behavioural differences to the controls 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. Living worms were assigned as adult (large complete worms) or offspring (small complete worms with regenerated body regions or incomplete worms), if possible. No mortality of worms was observed in the control and the limit test item concentration after exposure to the test item over 28 days. The total number of worms and thus the reproduction did not statistically significantly differ compared to the control. No evident changes in the worm behaviour or pathological symptoms were observed in the control as welf as in the limit test item concentration during the 28 days of exposure. At test end, the biomass dry weight of the worms was not statistically significantly different at the limit test item concentration 1000 mg/kg sediment dry weight compared to the control. Based on these findings the 28 -d NOEC is determined at 1000 mg/kg sediment dry weight.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater sediment:

1 000 mg/kg sediment dw