Reaction mass of 2,6-bis(1-phenylethyl)phenol and 2,4,6-tris(1-phenylethyl)phenol

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Due to observed mortality in the control group this study is considered invalid. However, the data obtained in this study are of scientific use and have been ascertained without any influence of the control group.

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
The test material is one of the constituents of the multi-constituent substance addressed by this registration dossier.

Radiolabelling:

no

Details on sampling:

SAMPLING FREQUENCY
- Sampling of test organisms: on days 0.5, 1, 2, 4, 6, 8, 14, 21, 22, 28, 35, 42 and 49 of the uptake phase as well as on days 0.5, 1, 2, 4, 6, 8, 14, 28, 41 and 58 of the depuration phase
- Sampling intervals/frequency for test medium samples: uptake phase days 0.5, 1, 2, 4, 6, 8, 13, 14, 16, 21, 22, 23, 28, 35, 37, 42, 49, 52 and on days 0.5, 1 and 2 of the depuration phase

SAMPLE STORAGE
- Water samples were stored at room temperature until start of analysis.
- Fish samples were directly analysed or stored in a freezer at -20 +/-2°C until the start of sample preparation

PREPARATION OF WATER SAMPLES
- Water samples: triplicate samples were prepared as follows:
1) Via enrichment. C8 cartridges were conditioned with 10 mL acetonitrile and 10 mL HPLC-water followed by loading with 150 mL of the sample. The analyte was eluted with 10 mL n-hexane. The resulting sample volume was reduced to approximate the half by a rotary evaporator and 1 mL acetonitrile was added before the evaporation continued to dryness. The residue was taken up in 2 steps with 2 mL of a 50:50 mixture of 0.1% aqueous ammonium formiate in acetonitrile by ultrasonic treatment for 2 x 15 seconds, transferred to a 5 mL measuring flask and filled up with the same solvent mixture. An enrichment factor of 50 was used to get sufficient amounts of tristyrylphenol for analysis.
2) Via standard addition. Additionally 5 replicates of water samples (5mL) were diluted 1:1 with 0.1% aqueous ammonium formiate in acetonitrile for stabilization. These solutions were fortified with 2, 4, 6, 8 and 10 µg/L test item followed by analysis via LC-MS/MS.
3) Stock solutions. Stock solutions from 2010-09-02 and 2010-09-10 of 20 mg/L test item in methanol were diluted with 0.1% ammonium formiate : acetonitrile (10 : 90) factor 500 (first factor 100 then factor 5) and analysed via LC/MS-MS

PREPARATION OF FISH SAMPLES
- Fish were homogenized in a centrifuge tube after 15 g Na2SO4 had been added. After adding 30 mL n-hexane, the resulting mixture was extracted, sonicated for 15 minutes at room temperature, shaken on a rotary shaker for 15 min. with 100 rpm and centrifuged at 3000 rpm for 5 min. Then, the supernatant was decanted into a 100 mL pear shape flask (which was weighed before). This extraction procedure was repeated twice with 20 mL n-hexane. The combined extracts were evaporated to dryness at 40°C at the rotary evaporator followed by determination of the lipid content. The residue was dissolved in a mixture of cyclohexane : ethyl acetate (50 : 50) and transferred quantitatively into a 25 mL measuring flask (5 x 5 mL of the mixture, followed by 10 seconds of sonication). 5 mL was purified by gel permeation chromatography (GPC) via liquid handler with a flow of 5 mL/min. Tristyrylphenol eluted in an interval of 30-37 min under the applied conditions. The eluate was transferred quantitatively with the mixture of cyclohexane : ethyl acetate (50 : 50) to a pear shape flask, evaporated to dryness followed by redissolving the residue in 5 mL acetonitrile and 15 seconds of sonication. The resulting sample was at least further diluted by factor 2 with a 10 : 90-mixture of 0.1% aqueous ammonium formiate : acetonitrile and analysed via LC-MS/MS.
- Determination of the lipid content. The fish were prepared as described above. The weighed pear shape flask was stored for 45 min at 105°C in a drying oven followed by cooling for 15 min in an exsiccator and weighed again.

Vehicle:

yes

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

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A flow-through exposure design was carried out. Membrane piston pumps provided the water flow-through. A precision syringe pump was used for the introduction of the stock solution.
- Chemical name of vehicle: metanol
- Stock solution: stock solutions of 20 mg/L were prepared with methanol.
- Concentration of vehicle in test medium: 0.1 mL/L

The use of a solvent was necessary to enable the application of the test item to the water streams. The test item has a very low solubility in water. Therefore, a preparation of stock solutions in water was not possible. Methanol (Roth, batch 697991, 691751 and 736941, purity >= 99.95%) was used for the preparation of stock solutions.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: rainbow trout
- Source: Forellenzucht Trostradt GbR, Dorfstr. 7, D-98646 Trostadt.
- Age at study initiation: 2 months
- Length at study initiation: 4.74 cm, SD 0.21 (control fish); 4.43 cm, SD 0.31 (test group)
- Weight at study initiation: 0.9887 g, SD 0.1117 (control fish); 0.7579 g, SD 0.2571 (test group)
- Weight at termination: 19.2207 g, SD 3.6323 (test group)
- Health status: No disease treatments were administered throughout the holding and testing.
- Description of housing/holding area: The animals were held at the testing facility at 13-17°C, diffuse light (0.1-10 µmol/m2/s, diurnal light with 16 h light / 8 h dark) and under flow-through conditions. The water exchange was 5 times the aquarium volume per day. The dissolved oxygen concentration was more than 80% of the air saturation value.

FEEDING DURING TEST
- Food type: Aller Performa Gr. 2, composed of fish products, oils and fats, cereal grains, oil seed products, byproducts, minerals and vitamins (Aller Aqua A/S, Allervei 130, DK-6070 Christiansfeld).
- Amount: 2% of body weight, adjusted weekly based on the weights of the sacrificed fish to account for growht during the experiment.
- Frequency: daily, as two feedings.

CLEANING
Uneaten food and faeces were siphoned from the test vessels two times daily (about 30 to 60 minutes after feeding). Care was taken not to injure the test organisms.

ACCLIMATION
Only rainbow trout with at least 12 days of acclimation and mortality < 5% within the last 7 days before the study started were used in the test. The stock population was fed the same type of food as during the test.

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

52 d

Total depuration duration:

58 d

Hardness:

10-250 mg CaCO3/L

Test temperature:

15 +/- 2°C
Due to technical problems the measured temperature fell below 13°C 4x for a few hours. The minimum temperature measured was 11.6°C.

pH:

6.0 - 8.5

Dissolved oxygen:

Not less than 60% of air saturation value.

TOC:

Mean value in 2µg/L vessel - uptake phase: 29.4 mg/L
Mean value in 2µg/L vessel - depuration phase: 0.30 mg/L
Mean value in control vessel: 1.4 mg/L
Determined two times before the uptake phase starts: -48 and -24 h and weekly thereafter (in all vessels).

Details on test conditions:

TEST SYSTEM
- Test vessel: glass aquaria with a water volume of about 200L and covered by glass tops
- Test volume: about 120L
- Aeration: gentle aeration was provided to prevent dissolved oxygen saturation dropped below 60% of air saturation value.
- Renewal rate of test solution (frequency/flow rate): a continuous water flow of 25L/h was provided, resulting in 5 water exchanges per day.
- No. of organisms per vessel: 100 fish per group
- No. of vessels per concentration (replicates): 1
- No. of vessels per control / vehicle control (replicates): 1
- Biomass loading rate: the nominal loading did not exceed a range of 0.1 to 1.0 g of fish (wet weight) per liter per day.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water of local origin. The water was filtered on activated charcoal and aerated for at least 24h to remove chlorine.

Nominal and measured concentrations:

Nominal concentration: 2 µg/L
Measured concentrations (enrichment method): mean measured concentration: 0.8 µg/L, SD 0.2, CV 31%
Measured concentrations (standard addition method): mean measured concentration: 1.3 µg/L, SD 0.5, CV 41%

Reference substance (positive control):

no

Details on estimation of bioconcentration:

Calculations and Statistics:

Growth rates (k growth): A linear correlation was calculated for the In (fish wet weight) vs. day.

Uptake / elimination rates of the test item: The uptake and elimination rate constants as a function of the total wet weights were determined as specified below:
The log concentrations in fish body weight were plotted versus time.

Uptake rates: For the calculation of a non linear regression the two phase exponential association model was used for Cf/Cw versus time:

Y=Y0+ SpanFast*(1-exp(-KFast*X)) + SpanSlow*(1-exp((-KSIow*X)) (Eq. 1)

With:
- SpanFast=(Plateau-Y0)*PercentFast*.01
- SpanSlow=(Plateau-Y0)*(100-PercentFast)*.01
- Kfast and Kslow are the two rate constant, expressed in reciprocal of the X axis time units. If X is in minutes, then K is expressed in inverse minutes.
- PercentFast is the fraction of the span (from Y0 to Plateau) accounted for by the faster of the two components

Depuration rates: A linear regression was done for the depuration phase data:

Y= intercept + slope . x (Eq. 2)

Kinetic bioconcentration factor: A kinetic BCF was not calculated because no first order uptake kinetic were observed.

Steady State bioconcentration factor: A BCF steady state was not calculated because no steady state was observed.

Bioconcentration factor at any time (BCFat) during the uptake phase: The concentration of test item in/on the fish or specified tissue thereof (Cf as ppm) divided by the concentration of the chemical in the surrounding medium (Cw as ppm).

Lipid normalization: Cf,L = [0.05 / L] . Cf (Eq. 3)

With:
- Cf,L = lipid-normalised concentration in fish (mg/kg wet weight)
- L = lipid fraction (based on wet weight)
- Cf = concentration of test substance in fish (mg/kg wet weight)

Software:
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.
- Excel, MICROSOFT CORPORATION
The data in this report were computer generated and rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor variations
may occur from these figures.

Lipid content:

2.6 %

Time point:

start of exposure

Remarks on result:

other: SD: +/- 0.4

Lipid content:

5.9 %

Time point:

end of exposure

Remarks on result:

other: SD: +/- 1.4

Lipid content:

8 %

Time point:

other: end of depuration

Remarks on result:

other: SD: +/- 1.2

Details on results:

Fish Observations, Mortaiity and non-lethal Effects:
On study day 34 100 % mortaiity occurred in the control group whereas the fish of the test item group did not show any abnormal symptoms. It was assumed that the reason for the mortaiity was a disease. Under the stereo microscope no visible symptoms of disease could be observed. On study day 35 four rainbow trout of the test item group were visually checked under a stereo microscope before determination of the test item analysis was carried out. The rainbow trout were considered to be free of parasites, protozoa, mycosis or any other disease indication. Since the study already lasted 34 days it was decided to proceed with the uptake phase of the test item fish (for details regarding validity please refer to part 8). A non significant mortaiity of 4 % was observed throughout the study in the test item group. No significant non lethal effects (morphological and behavioural) were found at the test item group and at the control group.

Feeding Behaviour:
No significant anomalous feeding behaviour was observed throughout the study in any one of the test groups (except day 33 in the control group).

Growth Rates:
Initial mean wet weight and length of the sampled fish of the control group was 0.847 g and 4.28 cm. At each sampling date wet weight and length of the sampled fish were determined. The mean wet weight of the test item group increased to 19.2 g, the mean length increased to 12.3 cm. On day 49 of the uptake phase the mean fish weight of the sampled fish was slightly lower. Trout populations always show some heterogeneity regarding fish size. This is due to the territorial behaviour and hierarchy trout always show when being kept in aquaria. This is why slight fluctuations in fish size occur and are acceptable. Weight data of the test item group were converted to natural logs and plotted vs. day. Linear least squares correlation was calculated. The growth rate was calculated as the slope of the linear correlation to be 0.0285.

Validity criteria fulfilled:

no

Conclusions:

This study is considered invalid due to the observed mortality in the control group. However, the study provides useful information on the behaviour of the test substance. Tristyrylphenol bioaccumulated in rainbow trout (whole) over a period of 49 days. A bioconcentration factor (BCF) steady state and a kinetic BCF were not calculated because no steady state and no first order uptake kinetic were observed. Therefore, BCF values (body weight based and lipid normalized based) have been calculated at any time measured during the uptake phase. With the TSP water concentrations determined via the enrichment method, which may overestimate the BCF values generated, the maximum BCF at any time was 8154 (body weight based, reached on day 35 of the uptake phase). It corresponds to a BCF lipid normalized of 6809. With TSP water concentrations determined via Standard addition method, BCF values were lower, i.e. the maximum value at anytime was 6116 (body weight based reached on day 35 of the uptake phase) and the BCF lipid normalized was 5106. Lipid normalized BCF have been calculated on base of 5 % lipid per fish.

Executive summary:

A bioaccumulation study with rainbow trout (Oncorhynchus mykiss) was conducted to determine the bioconcentration potential of tristyrylphenol (TSP). The study consisted of 2 phases: the exposure (uptake) phase and post-exposure (depuration) phase. A flow-through test with 2 groups was carried out: an untreated control group, and the test item with a nominal concentration of 2 µg/L was tested. The study was conducted over 111 days; the uptake phase lasted 52 days and the depuration phase lasted 58 days.

 

Analytical verification:

The concentrations of tristyrylphenol were determined via LC-MS/MS. 4 Fish samples of the test item group were sampled and analysed on days 0.5, 1, 2, 4, 6, 8, 14, 21, 22, 28, 35, 42 and 49 of the uptake phase as well as on days 0.5, 1, 2, 4, 6, 8, 14, 28, 41 and 58 of the depuration phase. Aqueous phase samples of the test item group have been taken and analysed from the uptake phase days 0.5, 1, 2, 4, 6, 8, 13, 14, 16, 21, 22, 23, 28, 35, 37, 42, 49, 52 and on days 0.5, 1 and 2 of the depuration phase. The concentrations of tristyrylphenol in the aqueous phase were measured via two different methods: the enrichment method and the standard addition method. The first method resulted in an overestimation of the BCF and therefore this has to be taken into account in the interpretation of the results. Two fish from the control group have been sampled and analysed on day 28 of the uptake phase. Aqueous samples from the control group have been sampled and analysed on day 0 and 28 of the uptake phase.

 

Evaluation:

The bioconcentration factors (at any time) have been calculated based on the fish body weight. No other bioconcentration factors (i.e. BCF kinetic and BCF steady state) have been calculated since the conditions for these calculations were not suitable. The uptake did not follow a first-order kinetic and a steady state was not observed.

 

Validity:

Due to observed mortality in the control group this study is considered invalid. However, the data obtained in this study are of scientific use and have been ascertained without any influence of the control group.

 

Results:

With the TSP water concentrations determined via the enrichment method, which may overestimate the BCF values generated, the maximum BCF at anytime was 8154 (body weight based, reached on day 35 of the uptake phase) and a BCF lipid normalized of 6809.

With TSP water concentrations determined via standard addition method, BCF values were lower, i.e. the maximum BCF at any time was 6116 (body weight based, reached on day 35 of the uptake phase) and a BCF lipid normalized of 5106.

Lipid normalized BCF have been calculated on base of 5% lipid per fish.

Reference 2

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 19/02/2008 (study initiation) to 04/09/2008 (date of the test report)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study performed according to the "fish dietary bioaccumulation study - Basic protocol - January 20, 2004" and the GLP. Although the guideline is not yet validated internationaly, the test procedure is in accordance with generally accepted scientific standards and described in sufficient detail.

Qualifier:

according to guideline

Guideline:

other: Fish, Dietary Bioaccumulation Study, Basic Protocol of January 20, 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Radiolabelling:

no

Details on sampling:

- Sampling intervals/frequency for test organisms: days 0 (only for control fish), 1, 5 and 10 of the uptake phase and on days 1, 2, 4, 7, 14, 28 and 42 of the depuration phase. Fish samples on day 10 (uptake phase) were analysed for content in guts (separate analysis).
- Sampling intervals/frequency for test medium samples: samples of the test and control diets were analysed for the test items at the beginning and during the uptake phase.
- Sample storage conditions before analysis: All samples were stored at -18 +/- 2 °C until start of analysis, if necessary.
- The lipid content was measured on each sampled fish.

Vehicle:

no

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

PREPARATION OF FOOD
Carrier : A standard fish food (floating and/or slow sinking pelletized diet) was employed. The food had an uniform pellet size to increase the efficiency of the feed exposure.
Preparation of food: 57.6 mg DSP and 86.4 mg TSP were added to 10 mL MTBE and 7.2 mL fish oil, gyrated on a vortex for 10 sec and added to 144 g of food in 750 µL portions (mixing by hand between addition of each portion). The maximum lipid content of the food did not exceed 20 % as a consequence of the spiking procedure. The spiked food was mixed on a bottle roller for 10 min and following shaken on a rotary shaker for 1 h at 20 rpm to assure homogeneity. MTBE was removed from the food under a gentle stream of nitrogen atmosphere for 24 h followed by shaking again the food for 30 min. Control food was analogously prepared but without addition of the test item. For the reference group 21 mg HCB and 10 mL MTBE were incubated for 30 min at 39 °C. 4.2 mL fish oil was added, gyrated on a vortex for 10 sec and added to 84 g of food in 750 µL portions (mixing by hand between addition of each portion). The maximum lipid content of the food did not exceed 20 % as a consequence of the spiking procedure. The spiked food was mixed on a bottle roller for 10 min and shaken on a rotary shaker for 1 h at 20 rpm to assure homogeneity. MTBE was removed from the food under a gentle stream of nitrogen atmosphere for 2 h followed by shaking again the food for 30 min. After 24 h the food was kept again under a nitrogen stream for 30 minutes shaken for further 30 minutes.
Contaminants : Feed analyses are maintained at the testing facility.
Storage conditions : the prepared food was stored under a nitrogen atmosphere at -18 ± 2 °C to prevent degradation.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name and Strain: Oncorhynchus mykiss (Rainbow trout), Gnathostoma, Pisces, Osteichthyes, Teleostei, Salmoniformes, Salmonidae
- Source: Forellenzucht Trostadt Gebr., Dorfstr. 7, D-98646 Trostadt/Thüringen. All fish used in the test originated from the same delivery of the supplier.
- Age at study initiation : juveniles, 3 month old
- Length at study initiation : mean of 10 fish = 5.80 cm
- Weight at study initiation : mean of 10 fish = 1.571 g
- Weight at termination : the mean weight of the control group increased to 4.8144g, the mean lenght increased to 7.76 cm.
- Method of breeding: Holding was performed at the test facility at 13 - 17 °C, diffuse light (0.1-10 µmol/m².s, diurnal light with 16 h light / 8 h dark were provided) and under flow-through conditions. The water exchange was at least 5 times the aquarium volume per day. The dissolved oxygen concentration was more than 80 % of the air saturation value.
- Feeding during test
- Food type: Aller 500 Gr. 2 (ALLER AQUA A/S, Allervej 130, DK-6070 Christiansfeld). The product is composed of fish products, oils and fats, cereal grains, oil seed products, by-products, minerals and vitamins.
- Amount: 2% of the initial fish weight
- Frequency: working daily.
During the test, fish were fed daily ad libitum at two feedings. The given food amount (approximately 3 %) was measured. Care was taken to ensure that fish have taken up most of the food presented. After 10 days of exposure, the fish were transferred to clean tanks and fed with untreated food for further 42 days. The amount of food was adjusted based on the weights of sacrificed fish to account for growth during the experiment.
Food ingestion rate: The ingestion rate is 0.03 g food / g fish / day.



ACCLIMATION
Only rainbow trout with at least 12 days of acclimation and mortality < 5 % within the last 7 days before the study started were used in the test. No disease treatments were administered throughout holding and testing. The stock population was fed of the same type of food which was used during the test.

Route of exposure:

feed

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

10 d

Total depuration duration:

42 d

Hardness:

In the control tank at test start (day1) = 57 mg CaCO3/L
In the control tank at test end (day52) = 55 mg CaCO3/L

Test temperature:

In the control group: from 15.4 to 16.4°C (mean = 16°C, SD +/-0.23°C)
In the test item group: from 15.3 to 16.5 °C (mean = 16.1°C, SD +/- 0.29°C)
In the HCB group: from 14.9 to 16.3 °C (mean = 15.9°C, SD +/- 0.34°C)

pH:

In the control group: from 7.06 to 7.70 (mean = 7.31, SD +/-0.16)
In the test item group: from 7.16 to 7.68 (mean = 7.34, SD +/- 0.14)
In the HCB group: from 7.08 to 7.67 (mean = 7.34, SD +/- 0.16)

Dissolved oxygen:

O2-saturation measured:
In the control group: from 75 to 100 % (mean = 88%, SD +/-6.2%)
In the test item group: from 78 to 100% (mean =91%, SD +/- 6.4%)
In the HCB group: from 72% to 100% (mean = 93%, SD +/- 8.5%)

TOC:

In the control tank: from 0.416 to 1.21 mg/L (mean = 0.686 mg/L, SD +/-0.298 mg/L)

Salinity:

Not applicable.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type : closed
- Material, size, headspace, fill volume: glass aquaria with a water volume of about 200 L, covered by glass tops. Test volumes = 110 L for the control and reference item group, 140 L for the test item group.
- Aeration: gentle aeration was provided
- Type of flow-through : Membrane piston pumps provided the water flow-through.
- Renewal rate of test solution : At least 3 renewals of dilution water per day were provided (425 L/d for the test item group, 330 L/d for the reference item group and the control group). The accuracy of the flow rates wsa checked weekly.
- No. of organisms per vessel: 125 fish per vessel for the control and reference item group, 155 fish for the test item group.
- No. of vessels per concentration (replicates): 1
- No. of vessels per control / vehicle control (replicates): 1
- Biomass loading rate: 0.59 g fish wet weight per liter per day for the control and reference item groups, 0.57 g fish wet weight per liter per day fish for the test item group.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water of local origin. the water was filtered on activated charcoal and aerated for at least 24h to remove chlorine.
- Holding medium different from test medium: no
- Intervals of water quality measurement:
pH-value, temperature and oxygen concentration: Twice per week in all test vessels. The water temperature in the control vessel was measured continuously (every hour) with a data logger.
Total hardness : at test start and end out of the control tank.
Total organic carbon (TOC) : Weekly from the water supply tank.
Residual chlorine : Weekly from the water supply tank. All measurement showed a concentration < 0.01 mg Chlorine/L.

OTHER TEST CONDITIONS
- Adjustment of pH: no data
- Photoperiod: diurnal light with 16 h light / 8 h dark
- Light intensity: 0.1 - 10 µmol photons/m².s

RANGE-FINDING / PRELIMINARY STUDY
no data

Nominal and measured concentrations:

Nominal concentration: 400 µg DSP/g, 600 µg TSP/g and 250 µg/g HCB
Mean measured DSP/TSP concentrations in food +/- SD, during the test : 342 +/- 50 µg DSP/g food and 509 +/- 110 µg TSP/g food.
Mean measured HCB concentrations in food +/- SD, at start of the test : 182 +/- 25.4 µg HCB/g food.
see tables 1 and 2 for single measured concentrations

Reference substance (positive control):

yes

Remarks:

Hexachlorobenzene (HCB), 99.7%, batch 313916/1, Sigma-Aldrich

Details on estimation of bioconcentration:

The elimination rate constant as a function of the total fish wet weight was determined. In addition, the assimilation efficiency (alpha), the biomagnification factor (BMF) and its lipid-normalised value (BMFL) were determined.

Lipid content:

5.8 %

Time point:

other: mean lipid content

Remarks on result:

other: lipid content of the fish in the DSP/TSP group

Lipid content:

5.78 %

Time point:

other: mean lipid content

Remarks on result:

other: lipid content in the fish in the HCB group

Type:

BMF

Value:

0.11

Basis:

whole body w.w.

Calculation basis:

kinetic

Remarks on result:

other: BMF calculated for TSP

Remarks:

Conc.in environment / dose:509 µg TSP/g food

Type:

BMF

Value:

0.355

Basis:

normalised lipid fraction

Calculation basis:

kinetic

Remarks on result:

other: BMF calculated for TSP considering a lipid factor of 0.309

Remarks:

Conc.in environment / dose:509 µg TSP/g food

Remarks on result:

other: DSP concentrations in fish were below the limit of quantification after end of uptake phase

Remarks:

Conc.in environment / dose:342 µg DSP/g food

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

18.4 d

Details on kinetic parameters:

For DSP : The course of the concentration in fish in dependence of uptake and depuration phase showed that there was no time and dosage related bioconcentration of DSP in the fish. The maximum concentration was reached on day one of the uptake phase and then decreased during the uptake phase. Finally, DSP concentrations were below the limit of quantification after end of uptake phase. Conclusively, DSP must be considered as not bioconcentrating under the test conditions. No uptake rate and depuration rate were determinable.

- Uptake rate constant (k1):
In contrast to DSP there was a clear uptake process for TSP. The uptake rate was determined to be 0.1052 *d-1 derived by the intercept of the linear regression.

- Depuration (loss) rate constant (k2):
For TSP, a distinct time related depuration was measured throughout the depuration phase providing the following results:
=> The slope of depuration was -0.0590 * d-1.
=> The intercept (= day 0 of depuration) was 2.725. The calculated concentration in fish was 15.3 µg TSP / g fish.
=> The mean measured concentration of TSP in fish on day 10 of the uptake phase was 21.6 ± 5.9 µg TSP / g fish.
=> The growth corrected depuration rate (kdepuration) was 0.038 d-1.
=> The half life time (t½) was 18.4 days.

- Assimilation efficiencies:
All evaluations are based on a food ingestion rate of 0.03 g food / g fish / day.
For TSP, the calculated assimilation efficiency ( alpha) was 0.138.

Metabolites:

No data.

Results with reference substance (positive control):

The biomagnification factor (BMF) was 1.35.
The lipid based biomagnification factor was 4.42 based on a lipid factor of 0.307.

- Uptake rate constant (k1):
A clear uptake process for HCB was also observed. The uptake rate was determined to be 0.2025*d-1

- Depuration (loss) rate constant (k2):
For HCB, a distinct time related depuration could be measured throughout the whole depuration phase of 42 days and gave the following results:
=> The slope of depuration was -0.0336 * d-1.
=> The intercept (=day 0 of depuration) was 3.1633. The calculated concentration in fish was 23.5 µg HCB / g fish.
=> The mean measured concentration of HCB in fish on day 10 of the uptake phase was 25.1± 6.2 µg HCB / g fish.
=> The growth corrected depuration rate (kdepuration) was 0.0112 d-1.
=> The half life time (t½) was 61.9 days.

- Assimilation efficiencies:
All evaluations are based on a food ingestion rate of 0.03 g food / g fish / day.
For HCB, the calculated assimilation efficiency ( alpha) was 0.507.

Details on results:

- Mortality of test organisms:
On study day 52 (day 42 of the depuration phase) one fish of the test item group and one in the HCB group died. No more mortality was observed at any other study day.

- Behavioural abnormalities:
No significant non lethal effects (morphological and behavioural) were found either at the test item, HCB or at the control group.

- Observations on body length and weight:
Weight data of the control and test item group were converted to natural logs and plotted vs. day. Linear least squares correlation was calculated. Growth rates were calculated as the slope of the linear correlation (see Table 3). The variances of the differences in the two slopes of the control and the test line were statistically compared by a student t-test. As there was no statistically significant difference, the test and control data were pooled for calculation of an overall fish growth rate (kGrowth) as the overall slope of the linear correlation.
Weight data of the reference item HCB were also converted to natural logs and plotted vs. day, linear least squares correlation calculated, growth rate calculated as the slope of the linear correlation (see Table 4) and statistically compared with the control. As there was no statistically significant difference, the reference and control data were pooled for calculation of an overall fish growth rate (kGrowth) as the overall slope of the linear correlation.

- Feeding behaviour:
No anomalous feeding behaviour was observed throughout the study in any one of the test groups. There were no food residues visible in the aquaria within 30-60 minutes after feeding.

- Validity criteria:
The following criteria for validity applied:
The temperature variation was less than +/- 2 °C
Oxygen saturation >=60 %
Mortality in the control and test groups were < 10 %
No significant concentrations of test chemical found in un-spiked food or control fish tissues
Observed bioaccumulation of the positive control: the assessed lipid based biomagnification factor was 4.40 which is within the range specified in the background protocol (0.77 – 4.70)

Table 3: Growth rates for the Control and Mixture of DSP / TSP Group   Control Mixtureof DSP / TSP Pooled Control +Mixtureof DSP / TSP n 110 110 220 Growth rate (= Slope) [d^-1] 0.0206 0.0220 0.0213 Stat. significance No -- Intercept 0.5924 0.5965 0.5945 CI 0.01651 – 0.02475 0.01857 – 0.02542 0.01868 – 0.02395 Table 4: Growth rates for the Control and HCB:   Control HCB Control + HCB n 110 110 220 Growth rate (= Slope) [d^-1] 0.0206 0.0242 0.0224 Stat. significance No -- Intercept 0.5924 0.6179 0.6052 CI 0.01651 – 0.02475 0.02109 – 0.02726 0.01983 – 0.02498 ANALYTICAL RESULTS OF DSP/TSP IN FISH: The mean concentrations (per sampling day) in fish are presented in Table 5. For DSP a decreasing average concentration was already observed during the uptake phase. After day 1 of the uptake phase an accumulation of the analyte was measured (5.89±1.92 µg/g fish). After day 10 of the uptake phase the average concentration of the analyte was<LOQ. For TSP increasing average concentrations were determined up to a final average concentration of 21.6±5.9 µg/g fish after the end of the uptake phase. Furthermore, the average concentration decreased during the depuration phase with the exception of day 7 to an average concentration of 1.61±1.07 µg/g fish. Additionally sampled fish from uptake day 10 were analysed for the concentration of DSP and TSP in gut. The results showed that there was no accumulation in the gut. Table 5: Mean concentrations of the Mixture of DSP / TSP in fish (n = 5 for control and n = 10 for test item group)    Concentrations (µg/g fish)       Study day   DSP (mean +/- SD)  TSP (mean +/- SD)  Control (mean +/- SD)  0  < LOQ  < LOQ  < LOQ  Uptake 1  5.89 +/- 1.92  7.39 +/- 2.84  < LOQ  Uptake 5  3.95 +/- 1.73  17.4 +/- 5.8  < LOQ  Uptake 10  < LOQ  21.6 +/- 5.9  < LOQ    gut < LOQ  gut < LOQ  < LOQ  Depuration 1  < LOQ  17.1 +/- 2.7  < LOQ  Depuration 2  < LOQ  14 +/- 6.8  < LOQ  Depuration 4  < LOQ  8.04 +/- 2.47(1)  < LOQ  Depuration 7  < LOQ  13.9 +/- 5.5  < LOQ  Depuration 14  < LOQ  6.71 +/- 2.09  < LOQ  Depuration 28  < LOQ  3.75 +/-1.93(2)  < LOQ  Depuration 42  < LOQ  1.61 +/- 1.07(2)  < LOQ (1)n = 9 due to elimination of 1 value by Grubbs outlier test (2)= for several fish the measured concentrations were < LOQ, nevertheless data were taken into account for calculation of the mean concentration. ANALYTICAL RESULTS OF DSP/TSP IN WATER  All measurements were below the LOQ. The lack of occurrence of the test item in the water phase showed that there were no losses to water and no other interferences that might have influenced the bioconcentration.  ANALYTICAL RESULTS OF HCB IN FISH The concentrations of HCB increased during the uptake phase. An average concentration of 25.1±6.2 µg/g fish was obtained at the end of the uptake phase. After depuration day 1 a slight increase in the accumulated HCB concentration compared to day 10 of uptake phase was observed but then the concentration decreased continuously to a final average concentration of 6.9±1.6 µg/g fish (see table 6). Table 6: Mean concentrations of HCB in fish (n = 5 for the control and n = 10 for the test item group)    Concentrations (µg/g fish)     Study day  HCB (mean +/- SD)  Control (mean +/- SD)  0  < LOQ  < LOQ  Uptake 1  3.4 +/- 1(1)  < LOQ  Uptake 5  16.5 +/- 3.8(2)  < LOQ  Uptake 10  25.1 +/- 6.2  < LOQ  Depuration 1  27.5 +/- 5.7  < LOQ  Depuration 2  24.9 +/- 7.2(3)  < LOQ  Depuration 4  23.2 +/- 6.7(4)  < LOQ  Depuration 7  15.8 +/- 4.6  < LOQ  Depuration 14  14.6 +/- 2.6  < LOQ  Depuration 28  7.7 +/- 2.1(4)  < LOQ  Depuration 42  6.9 +/- 1.6  < LOQ (1)= for several fish the measured concentrations were < LOQ, nevertheless data were taken into account for calculation of the mean concentration (2)= n = 9 due to preparation mistake (3)= n = 9 due to a fault injection (4)= n = 8 due to preparation mistake

Validity criteria fulfilled:

yes

Remarks:

validity criteria of the basic propotocol - January 2004

Conclusions:

DSP was not observed to bioaccumulate when applied via the diet. There was no bioconcentration measured for DSP. No bioconcentration factors were assessed.
TSP bioaccumulated in rainbow trout when applied via the diet, but also depurated when the application via the diet was stopped. The biomagnification factor was BMF = 0.110. The lipid based biomagnification factor was 0.355. Therefore, TSP is considered to have a low potential for biomagnification.

Executive summary:

A dietary bioaccumulation study was conducted to determine the half-life time (t_1/2, from the elimination rate constant, k_depuration), the assimilation efficiency (alpha), the biomagnification factor (BMF) and the lipid-normalised biomagnification factor (BMFL) of a Mixture of DSP / TSP, with rainbow trout (Oncorhynchus mykiss). The test items were administered to the test system via the diet. The study was performed according to the principles of the Basic Protocol (2004) and GLP.

 

A flow-through test with 3 tested groups was carried out. Fish were fed with spiked food during an uptake phase of 10 days followed by a depuration phase of 42 days. An untreated control group was tested. The test item Mixtureof DSP / TSP was tested with a concentration of 1000 µg/g food and hexachlorobenzene (HCB) was used as benchmark substance (= positive control) with a concentration of 250 µg/g food.The study was conducted over 52 days. All validity criteria required in the guideline were fulfilled.

 

Analytical verification:

The concentrations of distyrenated phenol (DSP) and tristyrenated phenol (TSP) were determined via LC-MS/MS, concentrations of HCB via GC-MS. 10 fish samples were taken from each tank at test start (day 0, only control fish) and on days 1, 5 and 10 of the uptake phase and on days 1, 2, 4, 7, 14, 28 and 42 of the depuration phase. Additionally, guts of 10 fish from day 10 (uptake phase) were analysed to investigate if the analytes remained in the gut after feeding.

Furthermore water samples, collected during uptake phase, were analysed to investigate if the analytes were present either by release from the fish via excretion or by release from the food during feeding.

DSP was not observed to bioaccumulate when applied via the diet. There was no bioconcentration measured for DSP. No bioconcentration factors were assessed. Furthermore analyses of the water samples demonstrated that DSP was absent conclusively ruling out that DSP was excreted by fish or released from the diet during feeding. Additionally, DSP was not detected in the gut samples.

TSP accumulated in rainbow trout when applied via the diet. The uptake rate was determined to be 0.1052 *d-1 derived by the intercept of the linear regression. During the depuration phase, the slope of depuration was -0.0590 * d-1, the intercept (= day 0 of depuration) was 2.725. The calculated concentration in fish was 15.3 µg TSP / g fish. The mean measured concentration of TSP in fish on day 10 of the uptake phase was 21.6 µg TSP / g fish. The growth corrected depuration rate (kdepuration) was 0.038 d-1 and the half life time (t½) was 18.4 days.

The biomagnification factor BMF was 0.110. The lipid based biomagnification factor was 0.355. TSP was present only in traces in the water samples and all determined concentrations were < LOQ. Analyses of guts revealed that TSP was absent.

HCB, being measured as a positive control, bioaccumulated as expected. The biomagnification factor BMF was 1.35. The lipid based biomagnification factor was 4.42.

The study revealed the following results :

	DSP	TSP	HCB
t1/2 (d)	n.a.	18.4	61.9
alpha	n.a.	0.138	0.507
BMF	n.a.	0.110	1.35
BMFL	n.a.	0.355	4.42

t1/2      =  Growth corrected half life

alpha   =  Chemical assimilation efficiency

BMF     =  Biomagnification factor

BMFL   =   Lipid normalised biomagnification factor

n.a.       =   Not applicable

Description of key information

TSP is found to be bioaccumulative (B) but not very bioaccumulative (vB) based on the following findings:

- BCF (kinetic, 5% lipid content) = ca. 2682 - 2706 L/kg ww

- BCF (kinetic, 5% lipid content, growth corrected) = 3972 L/kg ww

DSP is not bioaccumulative as it was found to be rapidly depurated.

MSP is not bioaccumulative based on its log Kow value being below the screening criterion of 4.5.

Additional information

A number of studies are available that address the bioaccumulation properties of 2,4,6-TSP. An independent evaluation of the available information has been carried out by Dr. Franck Gobas. The following information sources were included in his review:

1.    Flow-through bioconcentration test in Rainbow Trout (Scheerbaum, 2012)

2.    Dietary bioconcentration test in Rainbow Trout (Scheerbaum, 2008)

3.    In-vitro biotransformation assay of TSP in liver trout S9 or Rainbow trout hepatocytes

4.    QSAR based calculations (CAESAR)

5.    Environmental Risk Evaluation Report by Brooke et al, 2009.

The main conclusions of this assessment are summarized in the endpoint summary. For reasons of completeness, the full assessment report by Dr. Gobas is added an attachment.

1) Flow-through bioconcentration test in Rainbow Trout (Scheerbaum, 2012)

The test produced good quality data that are suitable for a bioaccumulation assessment of TSP. However, the interpretation of the test results included errors and significant short comings. Therefore, Dr. Gobas re-examined the data presented in this study, and derived the following parameters:

·      Uptake clearance rate k1 = 281 L/kg ww/d

·      Depuration rate constant kT = 0.088 d^-1

·      BCF (kinetic) = 3193 L/kg ww

·      BCF (kinetic, 5% lipid content) = 2706 L/kg ww

·      BCF (kinetic, 5% lipid, growth corrected) = 3972 L/kg ww

 

From the re-assessment of the data it can be concluded that 2,4,6-TSP is bioaccumulative (B), but not very bioaccumulative (vB), as the experimentally determined BCF values are > 2000 but < 5000.

 

2) Dietary bioconcentration test in Rainbow Trout (Scheerbaum, 2008)

The study by Scheerbaum (2008) provided a good interpretation of the results of the dietary bioaccumulation experiment. However, the study did not interpret the results of the experiment in terms of a bioconcentration factor of TSP in fish that can be used in a regulatory assessment. Dr. Gobas used recently developed methods for deriving bioconcentration factors from the results of the dietary bioaccumulation tests. Re-examination of the test results provided the following parameters:

·      Depuration rate constant kT = 0.059 d^-1

·      Half-life time t_1/2= approx. 12 d

·      Dietary uptake efficiency E_D= approx. 13 - 19%

·      BMF (lipid normalized) = 0.21 to 0.31 kg lipid/kg lipid

These results were used as input for the ADME-B calculator, a freely accessible excel calculation tool (Gobas et al, 2018), in order to derive a bioaccumulation profile and bioconcentration factor of TSP. The calculations indicate that the great majority of the ingested TSP (i.e. 72%) was biotransformed within the intestinal tract and an additional 5% of the ingested dose was biotransformed in the body of the fish. The reported lack of detectable TSP in the gut contents of fish is consistent with a high degree of biotransformation of TSP in the intestinal content.

The BCF of TSP calculated from the results of the dietary test was 3111 (± 380 SE) L/kg ww for the fish in the test and 2682 (± 327 SE) L/kg ww for fish with a 5% lipid content. The BCFs derived from the results of the dietary bioaccumulation tests are in line with the BCFs determined in the bioconcentration test, i.e. 3193 L/kg ww and 2706 L/kg ww for fish with a 5% lipid content.

From the re-assessment of the data it can be concluded that 2,4,6-TSP is bioaccumulative (B), but not very bioaccumulative (vB), as the experimentally determined BCF values are > 2000 but < 5000.

3) In-vitro biotransformation assay of TSP in liver trout S9 or Rainbow trout hepatocytes

In-vitro biotransformation assays of TSP in liver trout S9 or rainbow trout hepatocytes did not detect significant biotransformation rates of TSP. While in-vitro bioassays can provide useful information on biotransformation, the state-of-the-science of assessing the bioconcentration factor of very hydrophobic substances from the results of in-vitro bioassays is at an early stage. To date, a common finding in studies of the applicability of in-vitro biotransformation assays for the assessment of the bioconcentration factor is that in-vitro depletion rates underestimate in-vivo biotransformation rates and overestimate bioconcentration factors. This is likely the case for TSP as well. In-vivo studies indicate significant biotransformation rates, especially in the intestinal tract. Extrahepatic biotransformation is not captured by in-vitro bioassays that use fish liver S9 or hepatocytes. Other reasons for the inability of in-vitro bioassays to accurately assess in-vivo biotransformation rates include the selection of inappropriate concentrations of TSP in the in-vitro bioassay and difficulties generating aqueous solutions of extremely hydrophobic substances such a TSP.

 

4) QSAR based calculations (CAESAR)

QSAR based calculations using the CAESAR model generated estimates of the BCF for TSP between 2.5 and 250 L.kg-1. These BCF estimates for TSP are substantially smaller than the measured BCFs. QSARs are useful tools for the estimation of many properties, especially in absence of good quality empirical data. However, good quality bioconcentration and dietary bioaccumulation studies are available that can inform on the bioaccumulation behaviour of TSP.

 

5) Environmental Risk Evaluation Report by Brooke et al, 2009.

On behalf of the Environmental Agency of the United Kingdom, Brooke et al. (2009) reported calculations of the bioconcentration factor of TSP in fish and earth worms that are incorrect and do not agree with the experimental BCF of TSP in fish.

 

The calculations presented in Brooke et al. (2009) include four methodological errors. They include:

(i) The application of a growth-correction which violates the mass balance assumption that is the basis of the bioaccumulation model

(ii) The use of incorrect fish body weights in the calculation of the uptake rate constant of TSP in fish

(iii) Lack of accounting for the bioavailability of TSP in water and

(iv) The extrapolation of the relationship between the BCF in earthworms and KOW well beyond the domain of applicability (i.e. chemicals with a log KOW less than 6 according to Brooke et al. 2009).

 

When the methods for bioaccumulation assessment described in Brooke et al. (2009) are applied correctly, the following results are obtained:

·      BCF = 2046 L/kg ww

·      BCF (5% lipid content) = 1784 L/kg ww

·      BCF (growth-corrected) = 3177 L/kg ww

·      BCF (growth-corrected, 5% lipid content) = 2693 L/kg ww.

 

These calculated BCFs in fish are much closer to the experimentally determined BCFs than the calculated BCFs in Brooke et al. (2009) and indicate that TSP is a bioaccumulative (B) but not a very bioaccumulative (vB) substance in fish.

 

The estimate of the BCF of TSP in earthworms by Brooke et al. (2009) should not be included in the bioaccumulation assessment as the method for calculation is not appropriate for TSP and lacks any consideration of biotransformation.

 

Conclusion

It can be concluded from the data evaluated as part of this study that TSP does not meet the REACh criteria for very bioaccumulative (vB) substances. Both high-quality aqueous and dietary studies in juvenile rainbow trout show that the bioconcentration factor in fish with a lipid content of 5% is approximately 2700 to 2800 L/kg ww, which is well below the bioaccumulation criterion for very bioaccumulative substances (vB) of 5000.

The corresponding growth-corrected bioconcentration factor, which Dr. Gobas does not recommend for use in a bioaccumulation assessment due to violation of mass balance, is 3972 L/kg ww for a fish with a 5% lipid content and is also below the bioaccumulation criterion of very bioaccumulative substances (vB) of 5000.

 

In addition, TSP exhibits a lipid normalized biomagnification factor (BMFL) of 0.21 to 0.31 kg lipid/kg lipid, which is well below the de-facto criterion value of 1 kg lipid/kg lipid. This finding indicates that in aquatic food-webs, TSP can be expected to be subject to trophic dilution where concentrations of TSP in fish decline with increasing trophic level. Furthermore, the bioaccumulation profile of TSP as determined from the results of the aqueous and dietary studies in juvenile rainbow trout is distinctly different from that of HCB, which is a very bioaccumulative substance.

While TSP does not meet the REACh criteria for very bioaccumulative substances, it does meet the REACh criterion for bioaccumulative (B) substances because the bioconcentration factor of TSP is greater than the criterion value of 2000. TSP can be expected to bioconcentrate in fish due to its very hydrophobic properties (causing low elimination rates to water) and the modest capacity of fish to biotransform TSP in the body of the fish. However, due to TSP’s extreme hydrophobicity, bioconcentration can be expected to be a minor route of exposure of TSP to fish in the environment. Dietary uptake is the main pathway by which TSP is absorbed by fish. Efficient biotransformation of TSP in the intestinal tract of fish avoids biomagnification and prevents the build-up of high concentrations of TSP in organisms of aquatic food-webs. TSP is therefore not a substance that is very bioaccumulative in the environment.

In conclusion, the aqueous and dietary bioaccumulation studies by Scheerbaum (2008, 2012) are of good quality and provide the necessary information for a bioaccumulation assessment. The studies demonstrate that TSP is a bioaccumulative (B), but not a very bioaccumulative (VB) substance according to bioaccumulation criteria used in the EU.