1,1'-(methylenedi-4,1-phenylene)bis(3-butylurea)

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-02-26 to 2019-07-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

According to final decision on testing proposal (ECHA 2017).

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -III: Dietary Exposure Bioaccumulation Fish Test

Version / remarks:

2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.13 (Bioconcentration: Flow-through fish test)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1730 (Fish Bioconcentration Test)

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Details on sampling:

For sampling schedule please refer to table 1 in "any other information on materials and methods".

Any mortalities or abnormalities in appearance and behavior of the fish were recorded during the test.

The specific time points for each sampling refer to the start of each phase, which was initiated by feeding respective feed types. The uptake phase started with feeding spiked feed, the depuration with feeding unspiked feed. Here the first sampling was conducted 4 h after the first feeding with unspiked feed.

At each sampling event five fish from the treatment and the control vessel were removed, immediately rinsed in dilution water, blotted dry and then weighed and measured before being humanely killed. All samples were immediately frozen in liquid nitrogen and stored at <-18°C. Each fish was analyzed individually for the test item to generate five replicate data per sampling date. For the controls, the analysis of only one replicate sampled at the beginning (from the stock population) and end of the exposure phase, as well as at the end of the depuration phase were sufficient in case of obvious absence of background contamination. No additional fish were sampled from the stock population for chemical analysis during the study, as the fish sampled from the control chambers could be used to determine potential background levels of the test item.

For normalization matters, lipid contents of feed and fish were determined. Collected sample material was handled similarly to analytical samples (frozen in liquid nitrogen and stored at <-18°C). Triplicate samples of test and control diet were analysed for the lipid content at the beginning and end of the uptake phase.

Fish were sampled in triplicates from the treatment and the control at the end of the uptake period as well as the end of the depuration period for lipid analysis. Three additional reference fish were sampled from the stock population at the beginning of the test (see Table 1 in "any other information on materials and methods). All lipid samples were analysed for total lipid content according to a method by Smedes.

Vehicle:

yes

Remarks:

ethanol

Details on preparation of test solutions, spiked fish food or sediment:

PREPARATION OF SPIKED FISH FOOD
- Details on fish food: Commercially available trout feed pellets with a diameter of 0.8 or 1.1 mm (Inicio Plus, biomar, Denmark)

- Details of spiking:
The commercial feed Inicio Plus (biomar, Denmark) with a pellet size of 2 mm was used for experimentation (spiked and unspiked diets). An application solution was prepared taking into account the purity of 99.1 % of the test item: 50.5 mg HAT ISO were dissolved in 50.0 mL ethanol, corresponding to a concentration of 1.01 mg mL-1. For spiking, 250 g of fish diet were placed in a 2 L pear-shaped flask connected to a rotary evaporator equipped with a stainless steel capillary to apply the test solution via a solvent-inlet tube to the pellets under vacuum. 28.5 mL application solution (= 28.7 mg HAT ISO) was then spread among the feed particles through a spray application while mixing by rotation to ensure a homogenous distribution of test item on pellets. During spiking, a low pressure of approximately 550 mbar was set to run the application. Subsequently, the spiked pellets were dispersed on the bottom of an aluminum tray and left in the fume hood for 12 h in order to remove potential solvent residues before use. The control diet (for uptake) was prepared the exact same way, but without the test item in the spiking solvent.
The homogeneity and content of test diet was analysed after preparation. The stability of the test item on feed was also monitored after completion of the uptake phase.


- Test concentration: The test concentration was 100 mg kg-1 feed and was chosen based on the results of the non-GLP pre-test.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: rainbow trout

- Source: Fischzucht Störk, Wagenhausen 8, 88348 Bad Saulgau, Germany

- Age, length and weight at study initiation: The criteria of the test guideline were followed. Juvenile fish of similar weight and length (8 ± 4 cm) were selected for the test. Only healthy fish, free from observable diseases and abnormalities were used in the study. Mortality of the batch was less than 5% in the week preceding the start of the study.

- Description of housing/holding area:
The fish were held in water of the same quality as used in the test (purified drinking water) until the start of exposure. Test fish were held for at least 14 days prior to the test under equivalent water quality and illumination conditions to those proposed for use in the test. Fish were fed ad libitum throughout the holding period with trout food once daily.
De-chlorinated local tap water was used in accordance with the OECD guideline. The tap water is sourced from the Schmallenberg district water production plants, mostly fed by small springs and percolation. The purification process occurs on-site at Fraunhofer IME and includes filtration with activated charcoal, passage through a lime-stone column, and aeration to the point of oxygen saturation. To avoid copper contamination, plastic water pipes are used in the test facilities. The following water chemistry data, recorded monthly in the laboratory of the testing facility, were reported: temperature, pH, conductivity [μS cm-1], dissolved oxygen content [% ASV or mg O2 L-1], total residual chlorine [mg L-1], content of nitrate [mg L-1], nitrite [mg L-1], ammonium [mg L-1], phosphate [mg L-1], magnesium [mmol L-1], total hardness [mmol L-1], calcium hardness [mmol L-1], alkalinity [mmol L-1], NPOC content [mg L-1], metal content (cadmium, chromium, copper, iron, manganese, nickel, lead, and zinc) [μg L-1] and total chlorine [mg L-1].

- Feeding during test : The fish were fed daily with trout food Inicio Plus (0.8 or 1.1 mm, biomar, Denmark) at a level of 2.0 % of the body weight per day. Feeding was observed to ensure that fish are visibly consuming all of the food. The fish were dosed at approximately the same time on each day. Uneaten food and faeces were siphoned from the vessels within one hour after feeding. The daily feed ration was adjusted weekly.

- Food type: Commercially available trout feed pellets with a diameter of 0.8 or 1.1 mm (Inicio Plus, biomar, Denmark) were fed to the fish prior to the experimental phase. The same feed was used and prepared for the dietary exposure.


ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions: same as test

Route of exposure:

feed

Justification for method:

dietary exposure method used because stable, measurable water concentrations cannot be maintained

Remarks:

As outlined in detail under "Pre-Test Column Elution" in IUCLID Section 5.3.1 the aqueous route of exposure was found to be technically not feasible with the test item.

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

14 d

Total depuration duration:

4 d

Test temperature:

The water temperature in the aquaria ranged between 13.0 °C – 15.2 °C in both test vessels.

pH:

The pH in the test vessels ranged between 6.94 and 7.37.

Dissolved oxygen:

Oxygen saturations were between 73.7 and 101% in both vessels.

TOC:

Non-purgeable organic carbon (NPOC, as a measure of total organic carbon (TOC)) was determined between 0.782 and 1.204 mg L-1 in the test vessels. Thus, the concentration of organic carbon, which potentially originated from the test item, did not exceeded 10 mg L-1.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 L glass aquaria filled with 75 L water
- Type: open
- Aeration: yes (glass capillary)
- Type of flow-through: metering pump system
- Renewal rate of test solution: at least 15.6 L water per hour
- No. of organisms per vessel: 54 fish per vessel
- No. of vessels per concentration: 1
- No. of vessels per control: 1
- Biomass loading rate: A fish-to-water loading rate of 0.1 to 1.0 g fish (wet weight) per liter of water per day was applied.


OTHER TEST CONDITIONS
- Adjustment of pH: not applicable
- Photoperiod: 16 / 8 h light / dark cycle
- For OECD 305 part III (dietary exposure fish bioaccumulation), overall daily feeding rate used in the study: once a day, at the same time of the day

RANGE-FINDING / PRELIMINARY STUDY
- Test concentrations: 10, 100, and 1000 mg/kg feed
- Results used to determine the conditions for the definitive study: Please refer to RSS "Pre-Test Feed" in IUCLID Section 5.3.1

Nominal and measured concentrations:

Nominal: 100 mg test item/kg feed

Measured
Spiked feed at the start: mean concentration 117 mg/kg +/- 3.78 mg/kg
Spiked feed at the end: 100 mg/kg +/- 1.12 mg/kg

Reference substance (positive control):

no

Lipid content:

7.48 %

Time point:

end of exposure

Lipid content:

6.79 %

Time point:

other: end of depuration

Key result

Conc. / dose:

0.1 mg/g food

Temp.:

> 13 - < 15.2 °C

pH:

7

Type:

BMF

Value:

0.031 dimensionless

Basis:

other: kinetic BMF normalised for growth and lipid content

Key result

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

0.098 d

Remarks on result:

other: growth corrected

Key result

Rate constant:

growth rate constant (d-1)

Value:

0.034

Key result

Rate constant:

overall depuration rate constant (d-1)

Value:

7.11

Key result

Rate constant:

growth-corrected half-life (d)

Value:

0.098

Details on kinetic parameters:

- Depuration rate constant k2: 7.11 / d
- Indication of bi- or multiphasic kinetics: no

Metabolites:

n. a.

Results with reference substance (positive control):

n. a.

Details on results:

- Mortality of test organisms: there were no unscheduled mortalities in this study
- Behavioural abnormalities: none observed
- Observations on feeding behavior: there were no indications of palatability issues
- Observations on body length and weight: the animals showed a normal body weight and length development for their age and species
- Reproduction during test period: n. a.
- Other biological observations: no other effects observed
- Organ specific bioaccumulation: not investigated
- Bound residues forming a plateau: not observed
- Mortality and/or behavioural abnormalities of control: no
- Loss of test substance during test period: no
- Non-eliminated residues (NER) at the end of elimination phase: no
- Results with vehicle control: valid

Table 1 Summary of determined parameters for BMF calculation

Assimilation efficiency
	mg kg^-1	concentration at start of depuration in fish	1.10
	d^-1	depuration rate constant	7.11
	d	depuration uptake	14
	gfood*(gfish*d)^-1	food ingestion rate	0.02
	mg kg^-1	concentration in food	117
	unitless	Euler´s number	2.72
	unitless	assimilation efficiency	3.35
Growth corrected feeding rate
	gfood*(gfish*d)^-1	food ingestion rate	0.02
	gfish	mean weight fish start	8.27
	gfish	mean weight fish end	12.3
	gfood*(gfish*d)^-1	growth-corrected feeding rate	0.0135
Growth rate and depuration constant
	d^-1	growth rate constant	0.0342
	d^-1	growth rate corrected	7.08
substance-specific half-life
	d	substance-specific half-life	0.0975
	d	growth corrected substance-specific half-life	0.0979
Lipid correction factor
	%	lipid fraction in fish	7.14
	%	lipid fraction in food	23.1
	unitless	lipid correction factor	0.309
BMF results
	unitless	kinetic BMF	0.00942
	unitless	growth-corrected BMF	0.00947
	unitless	growth- and lipid-corrected BMF	0.0306

Validity criteria fulfilled:

yes

Conclusions:

According to this OECD 305-III compliant study, the resulting BMFKgL of 0.0306 indicates that the bioaccumulative potential of the test item is very low in Oncorhynchus mykiss when applied orally and uptake results from the digestive tract.

Executive summary:

A study was performed to determine the biomagnification factor of the test item in the fish species Oncorhynchus mykiss. The test item was administered to a test population of fish via food in accordance to the conditions of the OECD TG 305 for BMF studies. A test concentration of 117 ± 3.78 mg kg-1 test item was achieved by applying solvent-mediated spiking on regular feed pellets. In addition, solvent-spiked feed was used for a control population of fish to monitor potential mortalities or other adverse effects over time. All feed types were administered with a rate of 2 % mean body weight per day.

The test duration was 14 days for the uptake and another 4 days (96 h) for the depuration phase. During testing, no fish mortalities were recorded. Repeated samplings were done for both fish populations and five individuals were picked at each time point for chemical analyses. During the uptake phase, fish were sampled twice, at days 12 and 14. The highest mean tissue concentration was 1.33 ± 0.367 mg kg-1at day 14.

A depuration rate constant (k2g) of 7.08 was calculated (growth-corrected) from the elimination kinetics of the test item in fish matrix. This corresponds to a substance specific half-life (t1/2,g) of 0.0979 days (growth-corrected).

An assimilation efficiency (α) of 3.35 was determined by a test item concentration of C0,d =1.10 mg kg-1 in fish at the beginning of the depuration phase.

The kinetic BMFK was determined with 0.00942, which was corrected for growth-dilution effects resulting in a BMFKg = 0.00947.

Finally, a lipid correction of the BMF was conducted, in order to standardize the BMF for comparative analyses. The resulting BMFKgL of 0.0306 indicates that the bioaccumulative potential of the test item is very low in Oncorhynchus mykiss when applied orally and uptake results from the digestive tract.

Description of key information

Based on the results of a dietary biomagnification study in rainbow trout according to OECD 305, the test item revealed a kinetic BMF value of 0.00942 (corrected for growth and lipid: BMFkgl = 0.0306) This BMF value and its conversion into BCF values according to ECHA and OECD guidances demonstrate that the test item has no bioaccumulation potential. Please also refer to statement on bioaccumulation attached to IUCLID Section 13. For risk assessment purposes the worst case BCF value of 734.8 was used.

Key value for chemical safety assessment

BCF (aquatic species):

734.8 dimensionless

BMF in fish (dimensionless):

0.031

Additional information

OECD 305 III

A study was performed to determine the biomagnification factor of the test item in the fish species Oncorhynchus mykiss. The test item was administered to a test population of fish via food in accordance to the conditions of the OECD TG 305 for BMF studies. A test concentration of 117 ± 3.78 mg kg-1 test item was achieved by applying solvent-mediated spiking on regular feed pellets. In addition, solvent-spiked feed was used for a control population of fish to monitor potential mortalities or other adverse effects over time. All feed types were administered with a rate of 2 % mean body weight per day. The test duration was 14 days for the uptake and another 4 days (96 h) for the depuration phase. During testing, no fish mortalities were recorded. Repeated samplings were done for both fish populations and five individuals were picked at each time point for chemical analyses. During the uptake phase, fish were sampled twice, at days 12 and 14. The highest mean tissue concentration was 1.33 ± 0.367 mg kg-1at day 14.

A depuration rate constant (k2g) of 7.08 was calculated (growth-corrected) from the elimination kinetics of the test item in fish matrix. This corresponds to a substance specific half-life (t1/2,g) of 0.0979 days (growth-corrected).

An assimilation efficiency (α) of 3.35 was determined by a test item concentration of C0,d =1.10 mg kg-1 in fish at the beginning of the depuration phase. The kinetic BMFK was determined with 0.00942, which was corrected for growth-dilution effects resulting in a BMFKg = 0.00947. Finally, a lipid correction of the BMF was conducted, in order to standardize the BMF for comparative analyses.

The resulting BMFKgL of 0.0306 indicates that the bioaccumulative potential of the test item is very low in Oncorhynchus mykiss when applied orally and uptake results from the digestive tract.