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Bioaccumulation: aquatic / sediment

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Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Information on the category justification can be found in the Quaternary ammonium salts (QAS) category and section 13.2 of IUCLID.
Qualifier:
according to guideline
Guideline:
OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -III: Dietary Exposure Bioaccumulation Fish Test
Deviations:
not specified
Principles of method if other than guideline:
The biomagnification study was carried out following the principles of OECD TG 305 and were performed in accordance with the German animal welfare act under the Landesamt für Naturschutz, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Germany (permit 81-02.04.2018.A023).
GLP compliance:
not specified
Remarks:
Study assumed to be conducted following the best practices of GLP
Radiolabelling:
no
Details on sampling:
In the main study five animals of each group were sampled randomized on day 7 and day 14 of the uptake phase and after 10 h, 24 h, 2 days, 3 days, 7 days and 14 days of depuration. Samplings were done before feeding of the animals. After each sampling the remaining biomass of each group was determined by weighing the complete group of fishes to adjust the daily feed ration. The sampled animals were anesthetized in a water bath containing 150 mg/L MS 222 (Sigma Aldrich) and euthanized by a deep cut through the neck. Animals were weighed, blotted and the compartments (liver, gastrointestinal tract (GIT) and carcass) were dissected. In addition, the GIT was rinsed with ultra-pure water to remove remaining feed or feces presumably containing the test items. After weighing all compartments, the samples were shock frozen and stored at -20 °C until further sample processing for chemical analysis. The experimental diet was analysed before and after the exposure phase to verify the stability of the test items during the uptake phase.
Vehicle:
yes
Remarks:
methanol
Details on preparation of test solutions, spiked fish food or sediment:
Preparation of the experimental diets for pre and main study
For preparation of enriched test feed for the preliminary study commercially available fish feed pellets (Inicio Plus® BioMar, Denmark) with a size of 2 mm were spiked according to the method described by Goeritz et al. (2013). Feed batches prepared for the pre-study were spiked with one test substance each. The spiking procedure applied for the cationic test substance is described below. Specific informations related to the preparation of the test diets are summarized in below table.

An application solution of test substance was prepared taking the purity and the reduced molecular weight of the ion into account. Therefore, 23.5 mg test substance were dissolved in 20 mL methanol, corresponding to a concentration of 1 mg/mL. For spiking, 100 g fish feed 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. 3.44 mL application solution were applied to the feed particles by spray application, while the feed was thoroughly mixed by rotation to ensure a homogenous distribution of the test item on the pellets. During the spiking procedure, a low pressure of approximately 700 mbar was applied. After administration of the application solution 0.5 – 1.0 mL pure solvent was utilized to rinse the beaker of the application solution which was also applied via the spray-apparatus to ensure transfer of the whole amount of test item. Afterwards, a vacuum of 350 mbar was set to evaporate the solvent in the flask. In order to remove potential solvent residues, spiked pellets were dispersed in an aluminium tray and left in the fume hood overnight. Subsequently, spiked feed pellets were coated with sodium alginate and calcium chloride to avoid test item loss through leaching into test water. For this, a 2 % sodium alginate solution (w/w) was prepared by dissolving 3 g of sodium alginate in 150 g distilled ultra-pure water (UHQ). The suspension was heated to 100 °C and stirred until a homogenous viscous solution was obtained. 3.90 g of this solution were applied in a heated 1 L glass bottle and after even distribution of the sodium alginate solution on the inner walls of the bottle by gentle shaking, 100 g of the spiked feed pellets were added. The bottle was shaken thoroughly until no feed pellets were sticking to the wall anymore. In a last step, 0.89 g calcium chloride per 100 g feed were added and the bottle was mixed until no visual inhomogeneity could be observed.

During the main study, test substance was applied in pairs. The co-exposure of two test items reduced the number of separate studies and thus the required number of fish. The experimental diets were prepared and treated the same way as described above. Here, 250 g feed with a pellet size of 1.1 mm were prepared by solvent spiking. The application solution volumes of the ion pairs (Tetrabutylphosphonium bromide, TBP and test substance) were mixed beforehand and applied concurrently. For alginate coating, two batches of 100 g and one batch of 50 g feed were handled. The amount of sodium alginate and calcium chloride dihydrate applied to each batch were adjusted according to the protocol. The control diet used during the uptake phase for feeding control fish, was prepared in exactly the same way, but without the test substance in the spiking solvent. Homogeneity and content test substance in the spiked test diets were analysed directly after preparation in five replicates.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
Juvenile rainbow trout were purchased from Fischzucht Störk (Bad Saulgau, Germany) and maintained in a flow-through system in 200 to 250 L tanks filled with copper ion reduced tap water. Animals were kept under constant aeration at 14 ± 2 °C and under a 16:8 light-dark cycle until the start of the study for acclimatization purposes. The fish were fed with a commercially available food for fish breeding (Inicio Plus®, BioMar, Denmark).
Route of exposure:
feed
Justification for method:
dietary exposure method used because stable, measurable water concentrations cannot be maintained
Test type:
flow-through
Water / sediment media type:
other: copper reduced tap water
Total exposure / uptake duration:
ca. 14 d
Total depuration duration:
ca. 14 d
Details on test conditions:
Design of biomagnification studies with ionic test substance
A preliminary test was carried out before the main study to test the palatability and potential toxic effects of each experimental diet spiked with test substances. Treatment and control group, each consisting of 8 fishes, were tested during an uptake phase lasting 14 days. The feeding rate was increased from 2.0 to 2.5 % in the second week of the uptake phase. Experimental conditions during the pre-test were similar to the main study as described below. A single sampling was carried out at the end of the uptake phase. Two of the eight animals were shock frozen in whole and stored at -20 °C until chemical analysis. The other six animals were dissected and liver, GIT, filet and carcass of three animals were pooled to one replicate. The two pooled replicates per compartment were shock frozen and stored at -20 °C until analysis.

For the main study rainbow trout with an average weight of 5.42 ± 1.14 g (n = 160) were fed per treatment test diets enriched with two different ionic organic compounds, each. The first experimental diet was enriched with two cations (TBP and test substance). The resulting treatments and one control group (each 40 animals) were tested simultaneously. 80 L glass tanks were used as experimental tanks filled with 75 L of copper reduced tap water with a flow rate of 15.6 L/h resulting in a 5-fold water exchange per day. The condition of the test animals was examined with the aid of score sheets at least once per day. The water temperature, oxygen content (saturation in % and mg/L) and pH were checked every second day. Concentrations of nitrite, nitrate and ammonia were determined on day one, day seven and at the end of the tests.
The uptake phase of 14 days was followed by a depuration phase lasting 14 days. All animals were fed the non-spiked feed during the depuration phase. A feeding rate equivalent to 2 % of the body wet weight of the test animals per day was applied in compliance with the recommendation given by the feed manufacturer. Feces which may result in uptake via aqueous exposure, feces were removed at least three times per day to reduce the risk of a secondary exposure pathway via the water due to the release of freely dissolved test substance from feed residues and fecal matter.

Determination of content, homogeneity and stability of test substances in experimental diets
For the determination of substance concentrations in fish feed approximately 1 g feed was weighed into a 15 mL PP-vial. 4 mL MeOH were added, homogenized for 30 seconds and then treated in an ultrasonic bath for 10 min. The samples were centrifuged for 5 min at 5 000 rpm and the supernatants transferred into a volumetric flask. After homogenization, the dispersing tool was washed with 3 mL MeOH. This solution was then used for the second extraction step which is similar to the first one. A third extraction step (using the methanol for washing the dispersing tool) was performed and the combined supernatants were filled up with methanol to a volume of 10 mL. The extracts of the feed samples were diluted by a factor of 1000 in two steps yielding a concentration within the calibration range. For external calibration, calibration ranges of 0.1 - 20 μg/L for the cations were used. Here, at least 6 different calibration points containing blank matrix, test substance and MeOH were mixed. The amount of matrix in the calibration samples was equal to the amount of matrix in the samples. The specific concentrations measured in each sample were calculated based on the recorded weight.
The homogeneity of spiked feed was determined by measuring substance concentrations in five replicates. The stability of spiked feed was measured in triplicates after specific time points. For the preliminary study, the substance concentrations in the experimental diets were presented as the mean concentration measured at test start and test end. For the main study, the average feed concentration during the study was calculated by taking the mean of the concentrations measured at test start and at the end of the uptake phase.

Determination of test substance content in fish samples
The concentrations of the test substances in fish samples were determined by chemical analysis and all tissue concentrations were calculated based on a wet weight basis. For analysis, all tissue samples, except for liver and GIT, were homogenized first. Afterwards, approximately 1 g of homogenate was weighed into a 15 mL PP-vial and after addition of 4 mL MeOH mixed with a dispersing tool for 30 seconds. The whole liver and GIT samples were homogenized using a dispersing tool following addition of 4 mL MeOH. After mixing, samples were treated in an ultrasonic bath for 10 min, centrifuged for 5 min at 5,000 rpm and the supernatants were transferred into a volumetric flask. The dispersing tool was rinsed with 3 mL MeOH, which was then used to repeat the extraction step followed by a further rinse. Supernatants obtained from the three extraction steps were combined and filled up to a total volume of 10 mL. The extraction efficiency of the used extraction protocols was assessed. Therefore, blank matrices (GIT, liver and carcass) were spiked with a known amount of test substance:

- GIT: 1 μg/L for cations
- Liver: 2 μg/L for cations
- Carcass: 5 μg/L for cations

The extraction was performed as described above. For assessment, recovery rates were calculated. The matrix-matched calibrations with at least 6 points ranged between 0.05 - 20 μg/L for the cations. The extracts of the different matrices were diluted depending on the substance concentrations to yield a concentration within the calibration range. For the main study, TBP GIT samples from the uptake phase and 10 h and 24 h depuration phase were measured with a 1:5 dilution. Time points 48 h, 72 h, 7 d and 14 d of depuration of TBP GIT samples and all TBP liver and carcass samples were diluted 1:1. All test substance GIT samples were diluted 1:80 and all liver and all carcass samples 1:1. The organic content in each measured solution was between 50 - 55 %. For the calibration solutions, depending on the end volume (200 -1000 μL), 10 to 50 μL of the substance stock solutions were diluted with blank matrix, UHQ and MeOH leading to the approximate ratio of 1:1 (v:v) of organic solvent and UHQ. Importantly, depending on the dilution of the samples the amount of the matrix in the calibration solutions was adjusted.
Nominal and measured concentrations:
Mean content in feed samples [mg/kg]: 22.8 ± 0.27 (preliminary study), 23.6 ± 1.28 (main study)
Reference substance (positive control):
not specified
Details on estimation of bioconcentration:
Biomagnification and distribution factors were calculated based on the tissue concentrations measured at the end of the uptake phase. For calculation of steady-state biomagnification factors (BMFss) the substance concentration in the whole fish measured at the end of the uptake phase was divided by the concentration of the enriched feed. In case of the main study, the sum of the compartment concentrations was used to derive 'whole fish' concentrations. Individual tissue distribution factors of the different substances were calculated from the compartment-specific tissue concentration divided by the concentration in the whole fish.

Calculation of kinetic biomagnification factors of different matrices and of the whole fish (main study)
- The assimilation efficiency (α, absorption of the test item across the gut) was calculated.
- The feeding rate (I) used in the calculation was adjusted for fish growth to give an accurate assimilation efficiency (α). The growth-corrected feeding rate Ig was calculated.
- The kinetic BMF (BMFk) was calculated by multiplying the assimilation efficiency (α) with the feeding rate constant (I), divided by the product of the overall depuration rate constant (k2).
- The depuration rate constant (k2) was calculated by performing a linear regression of ln(concentration in fish) versus time [day].
- The growth-corrected biomagnification factor was calculated using the growth corrected depuration rate constant.

For more details on the calculation methods, kindly refer to the attached background section of the IUCLID.
Key result
Conc. / dose:
ca. 23.6 other: mg/kg in feed
Type:
BMF
Value:
ca. 0.027 other: g/g
Basis:
whole body w.w.
Calculation basis:
steady state
Key result
Conc. / dose:
ca. 23.6 other: mg/kg in feed
Type:
BMF
Value:
ca. 0.04 other: g/g
Basis:
whole body w.w.
Calculation basis:
kinetic
Key result
Conc. / dose:
ca. 23.6 other: mg/kg in feed
Type:
BMF
Value:
ca. 0.046 other: g/g
Basis:
whole body w.w.
Calculation basis:
kinetic, corrected for growth
Details on results:
Determination of homogeneity, content and stability of test substance on diet
For all test substances a linear relationship between the peak area of the quantifier mass trace and the concentration in the matrix-matched calibrations was found. The homogenous distribution of the test substance on feed was confirmed by the analysis of the test substance concentrations in five individually processed replicates. These measurements also represented the starting point of the stability measurement. The results of the homogeneity test of the two feed batches spiked with the two cations TBP and test substance. The relative standard deviations (RSD) were <10 % for both ions. Thus, a homogenous distribution of the test substances in the feed can be assumed.

All feed batches showed a homogenous distribution of the test substances. The stability measurements were performed to confirm stable concentrations during the whole uptake phase. Stability was defined as the recovery of a test substance in the spiked diet in comparison to the feed concentration measured immediately after feed preparation. In the preliminary test, recoveries of 96.1 – 136 % in comparison to the day of feed preparation were determined. During the main test the stability of the dietary concentrations of the test substances were assessed twice. First, 14 days after diet preparation (96.1 – 123% recovery) and second, at the end of the uptake phase, where recoveries of 86.2 – 109 % relative to the original concentrations were ascertained.

Based on the results of the stability measurements the dietary concentrations were calculated. The test diets had mean concentrations of 22.8 ± 0.27 mg/kg for the preliminary study and 23.6 ± 1.28 mg/kg during the main study for the test substance.

Biological Observation
No mortality or abnormal behaviour of the test animals were observed during the main study. The experimental diets were accepted by the test animals and showed a decent digestibility as confirmed by the texture and appearance of the feces. One fish in the “cation” was euthanized at day 25 due to injuries. The specific growth rates of the animals ranged from 1.95 to 2.71 %/d over the entire experiment. During the study, the feed conversion ratio (FCR) was 0.69 to 0.95.

Growth correction
Fish were measured and weighed at the beginning of the experiment as well as at respective sampling time points to monitor growth and associated growth-dilution effects during the feeding study. Growth of the test animals is also an important measure to detect potential adverse effects that may occur following dietary exposure. Growth rate constants were determined separately for the uptake and depuration phases, for the treatments and the control group, using the ln-transformed weights of the fish. A subsequent parallel line analysis (PLA, as suggested by the OECD Guideline) resulted in no statistical differences between the uptake and the depuration phase among the treated groups with P = 0.7784 for TBP/TMOA. No statistically significant difference was detected with regard to the growth of the treated groups (P = 0.7162). Hence it was deduced that neither adverse nor toxic effects were caused by the enriched diets. Weight data of the treatment groups could be pooled deriving the overall fish growth rate constant kg. The value determined for kg of 0.021 1/d, based on ln-transformed weight data of all fish, was then used for growth correction of the depuration constants.

Analysis of test substance content in fish samples
During the main study the extraction efficiency of the used extraction protocol was assessed by spiking samples with a known concentration in five replicates. Subsequently, tissue samples were extracted with the same protocol and recovery rates regarding the applied amount of test substance were determined. The estimated recovery rates were in a range of 80 - 120 % confirming the suitability of the applied extraction procedure. Note, standard deviations ranged from 5.0 - 28.0 %.

Calculation of substance specific tissue distribution and biomagnification factors in preliminary study
Based on the tisuue concentrations measured in fish collected on day 14 of the uptake phase (preliminary study), tissue distribution and magnification factors were calculated. The biomagnification factors was determined to be 0.028 for test substance.

Calculation of biomagnification factors (main study)
As steady state seemed to be reached after 14 days of exposure, steady state biomagnification factors (BMFss) could be calculated. Tissue concentrations measured during the uptake and depuration phase were used to calculate BMFk and BMFkg values for the different compartments and for the 'whole fish'. All derived kinetic BMF values are based on at least three time points measured during depuration, starting at day 0 of the depuration phase (day 14 of uptake phase). The calculated BMF values show that test substance in this study did not seem to biomagnify after dietary exposure. The BMFk and BMFkg were calculated for the test substance to be 0.040 and 0.046, respectively. The BMFk and BMFkg were 0.489 and 0.542 for test substance in the GIT. In general, the GIT and the liver showed the highest values for the BMFk and BMFkg. Growth corrected depuration rate constants k2g, were used to calculate BMF values. The absolute tissue concentrations during the uptake and depuration phase and all parameters that were used for the calculation of the BMF values.

Evaluation
The spiking procedure used for the enrichment of the experimental diets was suitable and led to stable and homogenous concentrations of the IOCs. The BMF values were determined as BMFss, BMFk and BMFkg. For the cations (TBP and Test substance) the values of the BMFkg were 1.7 times higher than the values of the BMFss. indicating that the steady-state conditions were not reached for the cation tissue concentrations at the end of the uptake phase. Neither the BMF values determined for the whole fish nor for any compartment indicate a significant biomagnification potential of the tested compounds in fish. Nevertheless, differences in the biomagnification potential of the different compounds were observed. Test substance led to BMF values that were 5 to 10 times higher than those of the other ions. In addition, a clear pattern of compartment or tissue specific magnification was observed. For all compounds the highest BMF values were determined for the GIT followed by the liver and finally the carcass. This might be explained by the location of the GIT and the transport of the compounds by blood via the vena portae from the GIT to the liver.

Interpretation of results
Overall, the measured BMF-values are low. The BMF for test substance was 0.0463 g/g. Overall, the study report authors concluded that the experimental BMF are low due to slow uptake and rapid depuration.

Conclusions for the assessment of ionic substances
The bioaccumulation of test substance was tested in an OECD 305 dietary uptake study. Test substance did not show distinct biomagnification (BMFkg < 0.1 g/g) and the highest tissue concentrations of test substance was found in the GIT. The results of this study showed that the uptake and elimination kinetics are decisive for the dietary BMF of the investigated test substance. The tissue analysis indicated that the low BMF of the investigated organic cations was due to a slow uptake from the GIT into the blood. This was also seen for some of the organic anions, but for those, the main reason for the low BMF was a rapid elimination, i.e. a high depuration rate. Hence, screening parameters primarily predicting adsorption, like Kfish/water, log Kow, log Dow, or the KHSA, may not be well suited to indicate high biomagnification following dietary uptake. Overall, it was concluded from the screening that ionization lowers the tendency of a chemical to bioaccumulate, compared to non-ionized chemicals. Aside of the well-known lipophobicity of ionized groups, fast depuration seems to be a major reason for the observed low biomagnification of ionic compounds, in particular anions. Fast depuration may happen due to rapid metabolism or conjugation of charged compounds, and future studies should test this hypothesis.

Results

Determination of homogeneity of cationic substances in fish feed used in pre and main study

 

Preliminary study

Main study

Content in feed samples [mg/kg]

Mean [mg/kg]

RSD

[%]

Content in feed samples [mg/kg]

Mean [mg/kg]

RSD

[%]

Test substance

20.96

22.5

4.3

25.16

25.9

4.4

23.62

26.98

23.46

27.35

22.04

24.26

22.36

25.81

 

Results of stability investigations of feed batches for pre and main study

 

 

Preliminary study

Main study

 

 

Test substance

 

tstart

t14 d

tend

tstart

t14 d

tend

mean conc. [mg/kg]

22.5

 

23.0

25.9

24.9

22.3

SD [mg/kg]

0.976

 

1.71

1.14

0.63

1.07

RSD [%]

4.34

 

7.43

4.41

2.54

4.81

mean recovery [%]

 

 

102

 

96.1

86.2

With tstart = day of preparation (here n = 5), t14 d = after 14 days (here n = 3), tend = end of the uptake phase (here n = 3), SD = standard deviation, RSD = relative standard deviations.

Feed conversion ratio (FCR) and specific growth rate (SGR) of experimental animals during the feeding study.

 

 

Uptake phase

(days 1 - 14)

Depuration phase

(days 15 - 28)

Total experiment

(days 1 - 28)

 

 

Test substance

Average body weight gain (g/fish)

2.39

2.37

4.76

Average feed intake (g/fish)

1.66

2.25

3.90

FCR

0.69

0.95

0.82

Wt1 (g)

5.18

7.57

5.18

Wt2 (g)

7.57

9.94

9.94

t2-t1 (d)a

14

14

28

SGR(%/d)

2.71

1.95

2.33

For accumulation: t1 = day 0 and t2 = day 14; for the depuration, t1 = day 14 and t2 = day 28, for the total experiment, t1 = day 0 and t2 = day 14. Wt1 = average body weight at t1; Wt2 = average body weight at t2.

Overview of extraction efficiencies of test substance

 

Compartment

Recovery

Test substance

GIT

83.2 ± 7.67 %

liver

102 ± 20.5 %

carcass

115 ± 8.61 %

 

Test substance concentrations in fish samples collected at end of uptake (pre and main study)

 

 

Preliminary study: Conc. ± SD [mg/kg]

Main study: Conc. ± SD [mg/kg]

Test substance

(calc.) whole fish

0.633 ± 0.042

0.6399

filet

0.206 ± 0.081

---

GIT

2.324 ± 0.799

8.2331 ± 1.661

liver

1.632 ± 0.389

1.0178 ± 0.190

carcass

1.218 ± 0.094

0.0862 ± 0.020

 

Overview of determined tissue specific BMFk and BMFkg

 

GIT

Liver

Carcass

Test substance

BMFk

BMFkg

BMFk

BMFkg

BMFk

BMFkg

0.489

0.542

0.0621

0.0833

0.00450

0.00742

 

Summary of 'whole fish' BMFk, BMFk and BMFkg

BMFk

BMFk

BMFkg

0.02709

0.04044

0.04629

 

Parameter for BMF calculation of test substance in compartments and 'whole fish'

 

Unit

 

GIT

Liver

Carcass

Fish

C0,d

mg/kg

conc. at start of dep.

12.104

1.129

0.0632

0.955

k2

1/day

dep. rate constant

0.222

0.0862

0.0557

0.173

t

day

duration uptake

14

14

14

14

I

gfeed/gfish * day

feed ingestion rate

0.02

0.02

0.02

0.02

Cfeed

mg/kgfeed

conc. in feed

25.9

25.9

25.9

25.9

α

 

 

assimilation efficiency

5.43

0.268

0.0125

0.350

kg

1/d

growth rate constant

0.0219

0.0219

0.0219

0.0219

k2g

1/d

growth rate corrected k2

0.200

0.0643

0.0338

0.151

t1/2

(uncorrected)

days

substance-specific half life

(based on uncorr. k2)

3.12

8.04

12.4

4.00

t1/2

(corrected)

days

substance-specific half life

(based on growth-corr. k2)

3.46

10.8

20.5

4.58

BMFk

 

 

kinetic BMF

0.489

0.0621

0.00450

0.0404

BMFkg

 

 

growth-corrected BMF

0.542

0.0833

0.00742

0.0463

For more details on the results, kindly refer to the attached background section of the IUCLID.

Validity criteria fulfilled:
not specified
Conclusions:
Under the study conditions, the source substance BMFss, BMFk and BMFkg values on whole body wet weight basis in rainbow trout were determined to be 0.02709, 0.0404 and 0.0463 g/g, respectively.
Executive summary:

A study was conducted to determine the biomagnification of the source substance, C18 TMAC (purity 95%), following the principles of OECD TG 305. For the main study rainbow trout with an average weight of 5.42 g were fed per treatment test diets enriched with source substance (23.6 mg/kg source substance in feed). The resulting treatment and one control group (each 40 animals) were tested simultaneously. The uptake phase of 14 days was followed by a depuration phase lasting 14 days. All animals were fed the non-spiked feed during the depuration phase. The concentrations of the source substances in fish samples were determined by chemical analysis and all tissue concentrations were calculated based on a wet weight basis. Chemical analysis of the source substances was performed by liquid chromatography with coupled mass spectrometry (LC-MS/MS). In the main study five animals of each group were sampled randomized on day 7 and day 14 of the uptake phase and after 10 h, 24 h, 2 days, 3 days, 7 days and 14 days of depuration. Biomagnification factor (BMF) and distribution factor were calculated based on the tissue concentrations measured at the end of the uptake phase. No mortality or abnormal behaviour of the test animals were observed during the main study. The experimental diets were accepted by the test animals and showed a decent digestibility as confirmed by the texture and appearance of the feces. One fish was euthanized at day 25 due to injuries. The specific growth rates of the animals ranged from 1.95 to 2.71 %/d over the entire experiment. During the study, the feed conversion ratio (FCR) was 0.69 to 0.95. Fish were measured and weighed at the beginning of the experiment as well as at respective sampling time points to monitor growth and associated growth-dilution effects during the feeding study. Growth rate constants were determined separately for the uptake and depuration phases, for the treatments and the control group, using the ln-transformed weights of the fish. A subsequent parallel line analysis (PLA, as suggested by the OECD Guideline) resulted in no statistical differences between the uptake and the depuration phase among the treated groups with source substance. No statistically significant difference was detected with regard to the growth of the treated groups. Hence it was deduced that neither adverse nor toxic effects were caused by the enriched diets. As steady state seemed to be reached after 14 days of exposure, steady state biomagnification factors (BMFss) could be calculated as 0.02709 g/g, which showed that source substance did not biomagnify after dietary exposure. In general, the GIT and the liver showed the highest values for the BMFk and BMFkg. The kinetic BMF (BMFk) and growth-corrected biomagnification factor (BMFkg) were calculated for the source substance to be 0.0404 and 0.0463, respectively. Overall, it was concluded from the screening that ionization lowers the tendency of a chemical to bioaccumulate, compared to non-ionized chemicals. Aside of the well-known lipophobicity of ionized groups, fast depuration seems to be a major reason for the observed low biomagnification of ionic compounds, in particular anions. Fast depuration may happen due to rapid metabolism or conjugation of charged compounds, and future studies should test this hypothesis. Under the study conditions, the source substance BMFss, BMFk and BMFkg values on whole body wet weight basis in rainbow trout were determined to be 0.02709, 0.0404 and 0.0463 g/g, respectively (Schlechtriem, 2021). Based on the results of the read across study, a similar low bioaccumulation potential is expected for the target substance.

Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Study period:
13 Jan 2020
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
QSAR prediction from a well-known and acknowledged tool. See below under "attached background material section" for methodology and QPRF
Qualifier:
according to guideline
Guideline:
other: REACH Guidance on QSARs: Chapter R.6. QSARs and grouping of chemicals
Principles of method if other than guideline:
The Bioconcentration factor (BCF) value was predicted for the test substance using BCFBAF v3.02 program of EPI Suite v 4.11. Since the test substance is an UVCB containing ionic and non-ionic constituents, the BCF values were predicted using regression-based and Arnot-Gobas BAF-BCF models respectively and using SMILES codes as the input parameter.
Justification for method:
other:
Details on estimation of bioconcentration:
The Bioconcentration factor (BCF) value was calculated for the test substance using BCFBAF v3.02 program of EPI Suite v 4.11. Since the test substance is an UVCB containing ionic and non-ionic constituents, the BCF values were predicted using regression-based and Arnot-Gobas BAF-BCF models respectively and using SMILES codes as the input parameter.
Key result
Type:
BCF
Value:
ca. 3.16 - ca. 162.4 other: L/Kg ww
Basis:
whole body w.w.
Calculation basis:
other: regression-based method and Arnot-gobas based method for ionic and non-ionic respectively
Remarks on result:
other: low bioaccumulation potential
Key result
Type:
BCF
Value:
70.06 other: L/Kg ww
Basis:
whole body w.w.
Remarks:
(weighted average value)
Calculation basis:
other: regression-based method and Arnot-gobas based method for ionic and non-ionic respectively
Remarks on result:
other: low bioaccumulation potential
Validity criteria fulfilled:
not applicable
Conclusions:
Using the regression-based and Arnot-gobas based methods of the BCFBAF v.3.02 program (EPI SuiteTM v4.11) for ionic and non-ionic constituents respectively, the BCF values were predicted to range from 3.16 to 162.4 L/kg ww (weighted average: 70.06 L/Kg ww).
Executive summary:

The Bioconcentration factor (BCF) value of test substance, TMAC T was predicted using regression-based and Arnot-Gobas BAF-BCF models of BCFBAF v3.02 program (EPI SuiteTMv4.11). The Arnot-Gobas method, takes into account mitigating factors, like growth dilution and metabolic biotransformations, therefore the BCF values using this method is considered to be more realistic or accurate. Therefore, except for ionic, pigments and dyes, perfluorinated substances, for which it is not recommended (as of now), the Arnot-Gobas method is used preferentially used for BCF predictions. Considering that the test substance is an UVCB containing majorly ionic (e.g., (e.g., the quaternary ammonium salts) and few non-ionic constituents (e.g., amines), the BCF values were predicted using regression-based and Arnot-Gobas BAF-BCF models respectively and using SMILES codes as the input parameter. The BCF values for the constituents ranged from 3.16 to 162.4 L/kg ww (log BCF: 0.50 to 2.21), indicating a low bioaccumulation potential. On comparing with domain descriptors, all constituents were found to meet the MW, log Kow and/or maximum number of correction factor instances domain criteria as defined in the BCFBAF user guide of EPISuite. Further, given that the major constituents are structurally very similar and vary only in the carbon chain length, a weighted average value, which takes into account the percentage of the constituent in the substance, has been considered to dampen the errors in predictions (if any). Therefore, the weighted average BCF value was calculated as 70.06 L/Kg ww (Log BCF = 1.85). Overall, considering either the individual BCF predictions for the constituents or the weighted average values, the test substance is expected to have a low bioaccumulation potential. However, taking into consideration the model’s training set and validation set statistics and the fact that the training set only contains 61 ionic compounds, the BCF predictions for the individual constituents are considered to be reliable with moderate confidence.

Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
2018 - 2019
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Information on the category justification can be found in the Quaternary ammonium salts (QAS) category and section 13.2 of IUCLID.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Divided over two mixtures, we exposed rainbow trout to water containing 10 alkyl amines and 2 quaternary alkylammonium surfactants for 7 days, analyzed different fish tissues for surfactant residues, and calculated the tissues’ contribution to fish body burden. Apparent BCFs (BCFapp) at the end of the 7- day exposure were calculated by dividing the surfactant body burden (including mucus) by the fish mass, and dividing this by the average measured concentration in water samples taken during the exposure phase. Studying chemical mixtures has the advantage that differences in behavior between chemicals are not obscured by biological variability or experimental variables. Bioconcentration studies with mixtures have been shown to provide similar results to studies with single chemicals.


Mixture 1: Nonylamine (P9), N,N-dimethyldecylamine (T10), Dodecylamine (P12), N,N-dimethyltridecylamine (T13), N,N,N-trimethyl-1-tetradecylammonium (Q14, i.e., C14 TMAC) and hexadecylamine (P16)
Mixture 2: N,N-dimethylnonylamine (T9), N,N,N-trimethyl-1-decylammonium (Q10, i.e., C10 TMAB), N-methyldodecylamine (S12), tridecylamine (P13), N,N-dimethyltetradecylamine (T14) and N-methylhexadecylamine (S16)
GLP compliance:
not specified
Remarks:
The study was conducted in research lab, therefore it was not mandatory to follow GLP.
Specific details on test material used for the study:
(a) Substance: N,N,N-trimethyl-1-decylammonium (Q10, CAS# 2082-84-0)
Purity: >98
SMILES: CCCCCCCCCC[N+](C)(C)C

(b) Substance: N,N,N-trimethyl-1-tetradecylammonium (Q14, CAS# 4574-04-3)
Purity: >98
SMILES: CCCCCCCCCCCCCC[N+](C)(C)C
Radiolabelling:
yes
Remarks:
Isotope labeled standards of Q10 (D21), Q14 (D29) were added to a portion of the extract corresponding to 12−75 mg of the sample.
Details on sampling:
Fish Exposure and Sampling.
The study was conducted with rainbow trout (Oncorhynchus mykiss) weighing between 98 and 165 g. The fish were purchased from Nordic trout Sweden AB and held in the aquaria facility prior to the experiment in February 2018. Ethical approval for the experiments was obtained from Stockholms djurförsöksetiskanämnd (decision 9967-2017). The experiments were conducted in 300 L fiberglass aquaria with a water renewal rate of 1.3 L min−1 (MIX 1) and 1.45 L min−1 (MIX 2). The water temperature was 10 °C and the pH 7.5. The water hardness was estimated to be 1.1 mM Ca2+ based on information from the water supplier. The aquaria water was circulated through a filter of polyester wool at 800−1200 L h−1 with an Eheim 2273 Pro 4+ pump. The outflow from the filter pump was placed above the water surface, providing for aeration. The lighting was dim and programmed on a 12 h light/12 h dark cycle. The fish were fed fish food pellets supplied by the fish farm at 1.0% of their body weight per day. No changes in fish behavior or appearance were observed during the experiment.

A solution of the test chemical mixture in methanol was infused continuously (3.5 and 3.8 μL min−1 for MIX 1 and MIX 2, respectively) into the water inflow using a syringe pump. The intended concentrations of the chemicals in water ranged from 2.5 to 50 μg L−1. They were selected with the help of modeled estimates of the BCF that were based on reported and extrapolated membrane water partition coefficients7 and biotransformation rate constants predicted with quantitative structure activity relationships (QSARs) to parameterize the BIONIC model. Based on these estimates, we selected an exposure concentration which would result in concentrations in fish that were clearly above the limit of quantification (LOQ) of the analytical method, while trying to keep the quantifiable exposure concentration as low as possible to minimize the risk of toxic effects and micelle formation.

For each mixture, the syringe pump was started in an aquarium containing no fish. After 16 h, to allow the concentrations to stabilize, 12 rainbow trout were added. After 7 d of exposure, the fish in the exposure aquaria as well as several unexposed (control) fish were sacrificed. Following stunning, blood was collected with a heparinized syringe from the caudal vein into a 2 mL Eppendorf tube. The fish were weighed and photographed, and the length was measured. The outer surface area of the fish was estimated from the fish weight according to O’Shea et al.

(surface area, in cm^2) = 11.2·(body weight, in g) ^ 0.65

The gill surface area was estimated from the fish weight according to the equation specific for O. mykiss from a report by Hughes,

(gill surface area, in cm^2) = 3.15·(body weight, in g) ^ 0.932

The surface of the fish posterior of the gills was rinsed with 100% methanol to remove test chemical residues adsorbed to the outer surface of the skin and absorbed in the skin mucus. Methanol is an effective solvent for extracting the test chemicals from tissue, as shown below. The fish were dissected and the liver, the kidney, the gills, and the remaining contents of the abdominal cavity were taken and weighed. Skin and muscle samples were prepared from the upper dorsal region on semi-frozen fish after the methanol rinse had removed the mucus. The main part of the subcutaneous fat tissue was included in the muscle samples rather than in the skin samples. All samples were stored frozen at −20 °C until further analysis.
Vehicle:
yes
Remarks:
methanol
Details on preparation of test solutions, spiked fish food or sediment:
The fish were fed fish food pellets supplied by the fish farm at 1.0% of their body weight per day. No changes in fish behavior or appearance were observed during the experiment.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
The study was conducted with rainbow trout (Oncorhynchus mykiss) weighing between 98 and 165 g. The fish were purchased from Nordic trout Sweden AB and held in the aquaria facility prior to the experiment in February 2018. Ethical approval for the experiments was obtained from Stockholms djurförsöksetiskanämnd (decision 9967-2017).
Route of exposure:
aqueous
Justification for method:
aqueous exposure method used for following reason:
Test type:
semi-static
Water / sediment media type:
other: water
Total exposure / uptake duration:
ca. 7 d
Hardness:
The water hardness was estimated to be 1.1 mM Ca2+ based on information from the water supplier.
Test temperature:
10 deg C
pH:
7.5
Details on test conditions:
Preparation of Fish Samples
For 6 fish from each aquarium and 3 control fish, samples of muscle, skin, liver, and gills were homogenized in a bullet blender (muscle and liver) (MiniG, SPEXsamplePrep) or in a cryo-mill (skin and gill) (Mixer mill cryomill, Retsch GmbH). A sub-sample of 0.5−1.2 g of the homogenate was extracted twice in methanol (4 mL, 50 °C, ultra-sound, 60 min), employing centrifugation at 4000 rpm for phase separation. Isotope labeled standards of Q10 (D21), Q14 (D29), and P16 (D33) were added to a portion of the extract corresponding to 12−75 mg of the sample. The extract volume was adjusted to 3 mL with methanol and then cleaned up on a weak cationic exchange SPE column (WCX, 60 mg, 30 μm particle size, waters). Solvent elution was performed by gravity flow. The column was conditioned with 2 mL of methanol, followed by 2 mL of Milli-Q water adjusted to pH 7 with ammonium hydroxide to activate the ion exchange. The sample extract was loaded onto the column, and it was subsequently rinsed with 20 mL of methanol. The analytes were released by first neutralizing the charged sites in the sorbent by the addition of 0.5% trifluoroacetic acid in Milli-Q water. The remaining water in the column was blown out with nitrogen. The surfactants were subsequently eluted from the WCX column with 0.65 mL of methanol. The remaining methanol was pushed out with nitrogen.

Whole blood was analyzed rather than plasma because of the small quantity of sample available and the anticipated low concentrations. Approximately 220 mg was transferred to a 13 mL polypropylene tube (Sarstedt AG & Co). After addition of internal standard, the blood was extracted with 3 mL of methanol using the same method employed for the other tissues. An aliquot of 1.5 mL of the combined extract was cleaned up on the WCX column using the same procedure employed for the other tissues, except that the column was rinsed with 10 mL (instead of 20 mL) after addition of the extract.

Water Samples
During the exposure experiments, water samples were collected. Special care was taken to minimize sample handling and in particular contact with surfaces during handling and to mix the water with solvent as quickly as possible in order to minimize sorption losses of test chemicals. Triplicate water samples were taken just before adding the fish, hourly for the first 8 h of exposure, and daily thereafter.

Aquarium water (600 μL) was sampled with an auto-pipette (polypropylene tip). The syringe was slowly filled and emptied five times before the sample was collected to avoid losses to the pipette tip inside surface. The sample was transferred to a 1.5 mL polypropylene sample vial containing 900 μL of methanol and isotope labeled standards of Q10, Q14, and P16. The water/methanol mixture (60 μL) was then analyzed using liquid chromatography with tandem mass spectrometry as described for fish.

The possibility of sorption losses to the vial was explored by spiking standard for 8 of the test substances into 0.5 mL of different water methanol mixtures in polypropylene vials and storing the vials at 4 °C overnight. For water/methanol ratios of 2:3 and 1:4, the recovery of all test analytes was >80%. The author noted that the strong tendency of the longer chained test chemicals to sorb to surfaces precluded the use of filtration or sorption-based methods to separate out the free (dissolved) fraction.

The methanol rinse of the fish surface (hereafter called “mucus”) was analyzed via direct injection after addition of internal standards.
Nominal and measured concentrations:
Q14 (N,N,N-trimethyl-1-tetradecylammonium) - 2.5 μg/L (intended) and 1.3 (time weighted mean measured) μg/L
Q10 (N,N,N-trimethyl-1-decylammonium) - 50 μg/L (intended) and 59 (time weighted mean measured) μg/L
Key result
Conc. / dose:
ca. 59 µg/L
Temp.:
ca. 10 °C
pH:
7.5
Type:
other: Apparent BCF (BCFapp)
Value:
ca. 0.1 L/kg
Basis:
whole body w.w.
Time of plateau:
7 d
Remarks on result:
other: Q10 (C10 TMAB) measured conc
Key result
Conc. / dose:
ca. 1.3 µg/L
Temp.:
ca. 10 °C
pH:
7.5
Type:
other: Apparent BCF (BCFapp)
Value:
ca. 31 L/kg
Basis:
whole body w.w.
Time of plateau:
7 d
Remarks on result:
other: Q14 (C14 TMAC) measured conc
Details on results:
Quality Assurance
The repeatability of the water method, measured as the average relative standard deviation of the triplicate samples collected at each time point, ranged between 1 and 11%. Poorer repeatability in a triplicate group was frequently associated with elevated concentrations of all of the more hydrophobic analytes in one of the triplicate samples. A plausible explanation is that a large particle of organic material (e.g., feces) had been collected with that particular sample. P16 could not be analyzed in the water samples due to an interference introduced by erroneous addition of an incorrect internal standard. The repeatability of the method for muscle and liver, measured as the relative standard deviation (RSD) of triplicate samples, varied from 2 to 29 and 1 to 12%, respectively. The LOQ was determined as mean + 10 × standard deviation of the concentrations measured in the tissues from the control fish. Because the concentrations in the exposed fish were not corrected for the concentrations in the control fish, a concentration below the LOQ in an exposed fish represents an upper limit of the concentration attributable to exposure. The concentrations in the exposed fish were well above the LOQ for most tissues. The major exceptions were Q10 and Q14, which were below the LOQ in muscle, liver, skin, and blood (Q10 only). P12 was also below the LOQ in three muscle samples. In this case, one control fish had much higher concentrations than the other three, which increased the LOQ by a factor of 7. This resulted in three of the six samples falling below the LOQ, although the six samples contained similar concentrations. Because of the uncertainty in the LOQ, these P12 data were included in the data analysis. The treatment of the data for Q10 and Q14 is described below.

Concentrations in Water
The mean measured concentrations in water ranged from 0.49 μg L−1 (T14) to 59 μg L−1 (Q10). The concentrations of P9, T9, T10, and Q10 were close to the intended concentrations, whereas the concentrations of the longer chained substances were markedly lower than the intended concentrations. The water concentrations reported here are bulk water concentrations, not freely dissolved water concentrations. In addition, a trend of decreasing concentrations over time by a maximum of 56% was observed for the longer chained substances. These observations can be explained by the stronger sorptive properties of the longer chained substances. Preliminary experiments suggested that the primary site of sorption in the aquarium system was the filter, and the increasing sorption over time could be related to the increasing accumulation of organic matter (e.g., fish excrement) on the filter over the course of the experiment.

Concentrations in Fish Tissue
The test chemical concentrations generally increased in the order muscle
Distribution Among Tissue Compartments
To calculate the quantity of the test chemical in the different tissues, the amount of each tissue in the fish was estimated and multiplied by the concentration in that tissue. The test chemical quantities in the different tissues were then summed to give the body burden in each fish. The total weight of the fish and the weight of the liver and gills were available from the dissection. For skin and mucus, the estimated outer surface area of the fish was used. We note that the distinction between mucus and skin is an operational one determined by the extraction method; mucus can be viewed as a more readily extracted fraction of the test chemical residues at the fish surface. The volume of blood was taken as 0.0495 mL g−1 fish. The remaining tissue (difference between the weight of the whole fish and the weight of liver, gills, skin, and blood) was assumed to have the concentration measured in the muscle. The contribution of the different tissues to the body burden is given for each fish, and the average contribution across all fish is plotted. All of the tissues, with the exception of blood, contribute at least 10% of the body burden for at least half of the test chemicals. There is no consistent negative correlation between the fraction of chemical associated with blood, muscle, and liver (tissues where the residue originates entirely from systemic uptake) and alkyl chain length (a measure of hydrophobicity). Thus, although adsorption to fish surfaces appears to play a significant role, its contribution to body burden cannot be simply predicted from surfactant hydrophobicity. In the following, we examine uptake in individual tissues.

Mucus
The presence of at least 10% of half of the test chemicals in the mucus compartment suggests that sorption of the cationic surfactants to the outer surfaces of fish can be nonnegligible. Surface area-normalized mucus−water distribu-tion coefficients (DMuc−W, in mL cm−2) calculated from the average concentrations in mucus and water show a strong positive relationship with alkyl chain length up to C14 for the ionizable alkylamines, with virtually no influence of the amine methylation. The large range in DMuc−W (2 orders of magnitude) indicates that sorption of the amines to mucus is not solely due to electrostatic attraction. In the linear alkyl chain length range of C9−C14, the increment per CH2 unit is 0.44 ± 0.03 (s.e.), which is higher than what was observed for sorption to soil organic matter (+0.28) but lower than that for sorption to phospholipid membranes (+0.59). S16 does not follow the same chain length relationship as the other ionizable amines; it lies at the same level as T14. The reason for this may be that the freely dissolved concentration was lower than the bulk concentration measured in the water samples, but such bioavailability issues for the most hydrophobic cationic surfactants were not studied in further detail here. The quaternary ammonium cation (QAC) Q14 has a 2.6 log units higher DMuc−W value than Q10. More remarkably, the DMuc−W values for the QACs were 360 and 37 times lower, respectively, than for the tertiary amines with the corresponding alkyl chain length. It is not clear what is causing the deviation between QACs and alkylamines. For both organic matter and phospholipids, lower affinities have been reported for QACs than analogue primary amines, in the order of 0.1−0.3 log units and 0.92−1.23 log units,7 for the respective sorbent materials. A model based on carbon chain length could allow for extrapolation of DMuc−W to other cationic surfactants, but the different behavior of S16 and the QACs indicates limitations to the applicability domain that need further study. Mucus solids in fish are predominantly glycoproteins. It is expected that overall the mucus is net negatively charged at near neutral pH because of the presence of substances such as sialic acid in oligosaccharide side chains. A cation-exchange capacity has been reported for mucus of ∼0.08 molC/g dry mucus. Hence, positively charged solutes may be electrostatically attracted into the mucus matrix and may also engage in electrostatic and/or hydrophobic interactions with the various charged biomolecules. Partitioning of cationic compounds between glycoproteins and water may therefore be partly driven by the same considerations underlying sorption to dissolved organic matter (mainly ion-exchange interactions) and phospholipids (i.e., a combination of electrostatic and hydrophobic interactions in hydrophobic pockets of the mucus). However, because of uncertainty in the actual mucus coverage on our fish, meaningful comparisons of the mucus sorption affinity with other solid materials cannot be undertaken.

Despite the apparently relatively strong sorption capacity of the thin outer mucus layer, it generally makes a small contribution to fish body burden because of the small body mass fraction of mucus. However, mucus may make a larger contribution to whole body burden when (i) the mucus to body weight ratio is higher (e.g., smaller fish, other fish species, other aquatic organisms), (ii) the surfactant is rapidly biotransformed, or (iii) the surfactant is not significantly taken up by the fish (as is apparently the case for the QACs). Gills. The gills are the tissue with the largest proportion of the body burden for P16, S16, T14, and Q14 in all individual fish and for P12 and P13 in four of five individual fish. Surface area-normalized gill−water distribution coefficients (DG−W) were calculated from the average concentrations measured in gills and water, and a calculated gill surface based on rainbow trout specific scaling factors. Surface area-normalized distribution coefficients for gills and mucus, DG−W and DMuc−W, follow the same trends and are of the same order of magnitude, with gill concentrations on average a factor of 3.9 higher. As with mucus, DG−W showed a strong positive relationship with alkyl chain length, but little influence of the head group for the amines. Also comparable to mucus, DG−W was much lower for the QACs Q10 and Q14 (a factor of 300 and 18, respectively) than for the tertiary amines of the same alkyl chain length. This could be related to the fact that fish gills are also coated in a mucus layer. Unlike the skin, the gills were not rinsed with methanol, so the gill samples included gill mucus. However, because the whole gill was extracted, some portion of the chemical residues in the gill samples may have been absorbed into gill tissue. The experimental protocol did not allow us to distinguish between the chemical sorbed to mucus and the chemical absorbed into the gill. The interior tissues (blood, liver, and muscle) accounted for more than half of the body burden of three of the amines (S12, T9, and T10) and more than a quarter of the body burden of the remaining amines. This indicates that gill uptake and distribution in the interior tissues are important for the bioaccumulation of alkyl amines in fish. The skin contained about 20% of the body burden of most of the amines.

Chemical residues could have reached the skin directly from water (via the mucus layer) or via the gills and internal circulation. The absence of quantifiable levels of Q10 and Q14 in any of the interior tissues (with the exception of Q14 in blood, which was just above the LOQ) suggests that the uptake of the QACs into interior tissue is very slow. In a study of the distribution of another QAC, hexadecylpyridinium bromide, in tadpoles following a 24 h exposure via water, concentrations in liver, kidney, and “fat body” were 30−100 times lower than the concentration in gills.

Tissue−Blood Distribution.
Blood is the transport medium for contaminants in fish, and the tissue distribution is governed by tissue−blood distribution coefficients. Of the tissues sampled in this study, tissue−blood distribution can be unambiguously studied for muscle and liver (for gills and skin, contact with water could have influenced the residue levels). Muscle−blood and liver−blood distribution coefficients (DMus−B and DL−B) were calculated as the quotients of the concentrations in the respective tissues and blood. This approach implies that there was an internal test chemical steady state in the fish. While there may not have been a steady state between the internal tissues and the external exposure medium, a near-steady state situation for chemical distribution among the major internal tissues is not an unreasonable assumption after 7 days of constant exposure. Muscle−blood distribution was much more uniform across the test chemicals than mucus−water and gill−water distribution. Examining the mean values of DMus−B for the six fish, the maximum (for S12) and minimum (for T9) differ by just a factor of 2.3. There is no consistent trend with chain length or methyl substitution of the amine group. This similarity in the DMus−B values suggests that the nature of the dominant sorbent is similar in blood and muscle, while the fact that the values are close to 1 suggests that the quantity of this dominant sorbent in the two tissues is similar. Given the sorption behavior of basic pharmaceuticals, phospholipids are expected to be the major sorbent for cationic surfactants in muscle tissue and potentially blood. Furthermore, the phospholipid content of rainbow trout muscle (1.65%) is similar to the lipid content of rainbow trout blood (1.4%), whereby the majority of the lipids in fish plasma are primarily in the form of lipoproteins, which may differ in sorptive capacity from phospholipids.

Compared to DMus−B, the mean values of DL−B vary more, ranging over a factor of 7. Furthermore, DL−B is clearly lower for the tertiary amines than for the primary and secondary amines. This suggests that the dominant sorbent phase and/or related considerations in the liver are different from those in blood (and by extension, muscle). Furthermore, the magnitude of DL−B (13−90) suggests that the quantity or the specific sorption capacity of the dominant sorptive phase in the liver is markedly higher than in blood, and the lower values for DMus−B (∼1) suggest that it is also markedly higher than in the muscle. Basic pharmaceuticals such as the selected serotonin reuptake inhibitors fluoxetine and sertraline, secondary amines with a pKa > 9.5 like the ionizable alkylamines studied here, accumulate to greater extents in the liver than in the muscle of fish exposed to sewage treatment plant effluents, but not in fish subjected to controlled exposure. Also amitriptyline, a phospholipophilic tertiary amine-based drug (pKa 9.8, log DMW 3.9)44 showed higher accumulation in fish liver than in muscle (gilt-head bream) in a controlled bioconcentration study, but the liver/plasma concentration ratios were not as high as the DL−B observed here. Higher values of DL−B compared to DMus−B have also been observed for chloroquine, a basic pharmaceutical, and attributed to greater lysosomal sequestration in liver cells, a process that has also been demonstrated for basic psychotropic drugs in slices of various rat tissues. However, given the similarity in dissociation constants (pKa), this phenomenon is insufficient to explain the differences in DL−B with respect to the amine substitution pattern observed in this study (i.e., T vs P, S amine). Further work is required to identify the nature of the dominant sorbent(s) and related considerations in the liver and assess the potential relevance for other internal organs/tissues.

The experimental data also allow estimation of the volume of distribution (Vd), an important parameter for extrapolating in vitro measures of e.g. metabolism to in vivo. In the context of quantitative in vitro−in vivo extrapolation for fish, Vd is defined as the quotient of the concentration in fish and the concentration in blood and describes the equivalent volume of blood that would contain the same amount of the chemical as 1 kg of fish.40 Vd was estimated using the residues that had been clearly taken up systemically (i.e., those in muscle, liver and blood). The mean Vd ranged from 0.49 L kg−1 for T9 to 1.49 L kg−1 for P13. This compares with plasma concentration based Vd values of other weak bases: 3.0 L kg−1 for diphenhydramine in fathead minnows47 and 0.32−0.48 and 0.19−0.28 L kg−1 for diphenhydramine and diltiazem, respectively, in killifish. These comparatively low values are an indication that a steady state for chemical distribution among internal tissues should be approached quickly. Vd was similar in magnitude to DMus−B.

Consequences for Bioaccumulation of Cationic Surfactants
Apparent BCFs (BCFapp) at the end of the 7-day exposure were calculated by dividing the surfactant body burden (including mucus) by the fish mass, and dividing this by the average measured concentration in water samples taken during the exposure phase. The BCFapp values ranged from 0.1 to 1260. Given the short exposure period, it is possible that the fish in this study had not approached steady state, in particular with respect to the distribution between water and internal tissues and between water and skin, and that the steady-state BCFs are higher. The proximity of some of the BCFapp values to the regulatory threshold for PBT chemicals in REACH (a BCF of 2000) indicates that further study of the bioaccumulative properties of cationic surfactants is warranted, whereby the longer chained alkyl amines appear to be the most bioaccumulative. The successful recoveries of a wide range of cationic surfactants from both water and fish tissues samples demonstrate that bioconcentration studies with fish with cationic surfactants are feasible. We note that some improvements in the methodology would be helpful, particularly with respect to the stability of the aqueous exposure concentration of the longest chain surfactants. Working with mixtures of analogue cationic surfactants allowed for consistent evaluation of differences in accumulation trends due to structural features. The tissue distribution results raise a number of questions regarding the assessment of bioaccumulation of cationic surfactants. If we presume that much of the test chemical in the gills did not enter the internal circulation system, then a significant fraction of the test chemical present in the whole fish was not able to reach other target tissues and thus was constrained in its ability to exert adverse effects on the fish. This is most apparent for Q14, P16, and S16, for which at least 50% of the body burden was present in gills and mucus. Similarly, much of the chemical residue in the fish was not present in tissues that would normally be subject to human consumption [e.g., Q10 and Q14 were not present in muscle above the LOQ, and for the primary amines the category muscle (which included other tissues) contributed at most 18% to the body burden]. Consequently, humans eating just the muscle of a fish would be exposed to much lower levels of these chemicals than predators eating the same fish. This suggests that edible tissue analysis could be more appropriate than whole fish analysis for human exposure assessment. Whole fish analysis would be relevant from the perspective of biomagnification and ecological exposure assessment or if adverse effects result from adsorption to epithelial tissues (e.g., the gills). To be relevant for biomagnification, there must be efficient dietary uptake of the chemical. Still, predicted steadystate BCFs based on experimental phospholipid−water distribution coefficients suggest that QACs should accumulate to a similar extent as ionizable analogue amines. Prolonged exposure studies with smaller sized fish, including adequate uptake and elimination phases, are the logical next step to better assess whether bioconcentration of permanently charged surfactants is mainly limited kinetically and to further improve parameterization of models to predict BCF and toxicokinetics for permanently charged compounds.

Results

Table: Concentrations (µg L-1) from replicate analyses of MIX 1 substances in amuscle sample

 

P9

T10

P12

T13

Q14

P16

Sub-sample a

16

160

343

338

3.4

83

Sub-sample b

19

207

407

435

4.1

82

Sub-sample c

18

202

404

422

2.2

79

Mean

18

190

385

398

3.2

81

RSD

0.10

0.14

0.09

0.13

0.29

0.02

 

Table: Concentrations (µg L-1) from replicate analyses of MIX 2 substances in aliver sample

 

T9

Q10

S12

P13

T14

S16

Sub-sample a

3082

7.5

21545

22782

3079

9714

Sub-sample b

2910

8.2

21788

24366

3291

9684

Sub-sample c

3127

9.6

21114

22676

3144

9534

Mean

3040

8.5

21482

23275

3171

9644

RSD

0.04

0.12

0.02

0.04

0.03

0.01


 

Table: Concentrations (ng g-1) of test chemicals in blood from control fish and exposed fish.

 

P9

P12

P13

P16

 

S12

S16

 

T9

T10

T13

T14

 

Q10

Q14

Control fish

#1

 

2.0

 

3.4

 

2.1

1.1

 

 

 

1.0

 

 

1.3

0.3

#2

 

 

 

3.9

2.5

 

 

 

1.0

 

1.5

0.3

#3

 

 

 

3.4

1.8

 

 

1.5

 

 

1.8

0.4

#4

 

 

1.3

2.1

1.5

 

 

 

 

 

0.7

0.1

#5

 

3.0

 

1.5

1.1

 

2.0

 

 

 

0.8

0.2

LOQ

 

7.4

 

13.0

7.1

 

 

 

5.4

 

5.7

1.4

Exposed fish#

#1

31.9

558

495

119

 

1245

264

 

305

612

890

309

 

0.7

2.0

#2

64.7

941

566

133

1001

279

334

454

816

266

0.9

1.8

#3

58.3

917

597

140

1110

304

475

531

1018

276

0.8

1.9

#4

33.7

624

533

120

1021

290

251

366

858

378

1.4

1.3

#5

39.5

583

590

97

1181

340

323

389

795

304

0.8

1.6

#6

49.1

982

434

171

996

304

291

606

1205

270

1.5

1.7

Mean

46

767

536

130

1093

297

330

493

930

301

1.0

1.7

RSD

29%

26%

12%

19%

10%

9%

23%

22%

17%

14%

34%

13%

Italics = <LOQ

#Since the test chemicals were exposed in two mixtures, each fish # shows the results for 2 individuals, 6 test chemicals in each


 Table: Concentrations (ng g-1) of test chemicals in muscle from control fish and exposed fish.

 

P9

P12

P13

P16

 

S12

S16

 

T9

T10

T13

T14

 

Q10

Q14

Control fish#

#1

 

129

11

1.90

 

14

14.3

 

14

5.89

10

20

 

8.2

2.75

#2

 

24

5.3

1.10

7

9.0

11

4.36

4.2

8

2.3

1.93

#3

 

15

4.5

0.66

6

6.6

8.3

1.11

1.3

7

2.5

2.61

#6

 

17

4.7

0.58

5

3.7

8.1

1.80

1.4

11

2.9

2.81

LOQ

 

600

35.8

7.12

50

53.3

39.4

25.6

44.7

68

32.3

6.57

Exposed fish#

#1

11.6

409

451

90

 

1384

313

 

173

459

580

204

 

1.1

4.90

#2

43.5

780

527

117

1101

322

173

313

593

160

8.6

3.92

#3

29.6

597

654

120

1319

367

200

292

601

161

2.4

2.83

#4

31.0

677

514

152

1228

249

146

347

882

203

1.1

3.13

#5

28.6

847

596

98

1267

347

158

357

767

194

2.3

3.29

#6

20.2

449

426

86

1052

317

111

300

606

188

0.9

2.36

Mean

27

626

528

111

1225

319

160

345

672

185

3

3

RSD

39%

28%

16%

22%

10%

13%

19%

18%

19%

11%

108%

26%

#Since the test chemicals were exposed in two mixtures, each fish # shows the results for 2 individuals, 6 test chemicals in each

Italics = <LOQ


Table: Concentrations (ng g-1) of test chemicals in liver from control fish and exposed fish.

 

P9

P12

P13

P16

 

S12

S16

 

T9

T10

T13

T14

 

Q10

Q14

Control fish#

#1

22

58

3

141

 

5.3

1.8

 

11.1

45

88

43

 

2.1

117

#2

 

21

15

55

10.8

3.5

7.4

13

37

9

2.3

60

#3

 

13.9

3

29

6.6

2.4

7.8

7.1

12

22

4.1

25

#6

 

8.2

3

24

6.1

2.0

7.7

7.0

8

45

1.7

16

#7

 

7.7

3

16

8.7

2.0

8.5

6.4

7

5

4.0

18

LOQ

 

230

58

567

30

9.3

24

182

375

210

14.3

477

Exposed fish#

#1

1011

35571

57540

5446

 

65630

22374

 

3588

4791

6570

5131

 

14

206

#2

2749

50242

49595

5714

40796

16314

2592

7207

8202

3165

20

146

#3

2258

50095

24376

6162

29036

10296

2863

11390

13696

2458

12

116

#4

1709

37024

43753

4785

40791

15915

1593

15685

20532

3531

20

111

#5

1410

38611

52619

4982

59171

19765

5463

8660

12097

5179

11

166

#6

1124

42593

54825

5850

61662

19043

7230

6254

9499

6424

11

147

Mean

1710

42356

49676

5490

52560

18120

3887

8998

11766

4530

15

149

RSD

40%

15%

26%

10%

30%

24%

49%

44%

43%

33%

25%

23%

#Since the test chemicals were exposed in two mixtures, each fish # shows the results for 2 individuals, 6 test chemicals in each

Italics = <LOQ


 

Table: Concentrations of test chemicals in gills from control fish and exposed fish.

 

P9

P12

P13

P16

 

S12

S16

 

T9

T10

T13

T14

 

Q10

Q14

Control fish#(ng g-1)

#1

 

1.8

4.1

1.51

 

3

0.69

 

18.2

3.8

0.34

1.74

 

1.6

9.87

#2

 

0.6

6.4

2.01

4

0.85

5.2

1.9

0.87

3.73

0.7

2.09

#3

 

2.0

4.9

52

81

3.59

3.0

1.6

26

19.28

0.6

1.86

LOQ

 

8.6

16.4

310

480

18

91

14.5

158

104

6.6

50

 

Exposed fish#(ng g-1)

 

#1

1045

18468

15969

7965

 

15191

19962

 

1126

2466

9077

6248

 

25.7

831

 

#2

1364

20381

17905

9190

16811

21384

1320

2314

9391

5753

24.5

603

 

#3

ND1

ND1

17705

ND1

16976

22341

1432

ND1

ND1

5824

31.0

ND1

 

#4

1131

17880

20913

6892

18286

26208

1130

2250

9586

7651

29.4

553

 

#5

1347

22031

19295

9192

16009

24620

1240

2385

11785

6627

28.9

705

 

#6

1111

22396

14984

10240

14911

20464

869

2453

11130

5408

25.8

656

 

Mean

1199

20231

17795

8696

16364

22496

1186

2374

10194

6252

28

669

 

RSD

12%

10%

12%

15%

8%

11%

16%

4%

12%

13%

9%

16%

Exposed fish#(ng cm-2)

#1

16.2

286

222

123

 

211

278

 

15.7

38

140

87

 

0.36

12.9

#2

22

326

235

147

221

281

17.4

37

150

76

0.32

9.6

#3

ND1

ND1

ND2

ND1

ND2

ND2

ND2

ND1

ND1

ND2

ND2

ND1

#4

21

331

324

128

283

406

17.5

42

178

119

0.46

10.2

#5

24

391

275

163

228

350

17.6

42

209

94

0.41

12.5

#6

14.8

297

195

136

194

266

11.3

33

148

70

0.34

8.7

Mean

19.5

326

250

139

228

316

15.9

38

165

89

0.38

10.8

RSD

20%

13%

20%

12%

15%

19%

17%

10%

17%

21%

15%

17%

#Since the test chemicals were exposed in two mixtures, each fish # shows the results for 2 individuals, 6 test chemicals in each ND1: No data due to instrumental problems

ND2: Missing dissection weight


 

Table: Concentrations of test chemicals in skin from control fish and exposed fish.

 

P9

P12

P13

P16

 

S12

S16

 

T9

T10

T13

T14

 

Q10

Q14

Control fish#(ng g-1)

#1

1.1

3.9

14

4.1

 

18.4

8.5

 

2.2

2.0

0.5

4

 

2.0

2.7

#3

4.58

2.9

9

2.1

10.6

4.9

3.2

2.9

1.4

2

2.1

7.6

#6

1.36

4.7

13

 

15.7

4.4

2.7

1.3

2.2

3

2.4

3.4

LOQ

22

12.8

39

16.7

54

29

7.4

10.0

9.7

12

4.1

31

Exposed fish#(ng g-1)

#1

70

2716

2037

364

 

4457

970

 

660

699

1879

1000

 

17

20

#2

167

4633

2161

415

2569

942

322

803

1877

615

3.7

17

#3

164

4372

2132

423

2398

927

301

786

2115

597

3.8

15

#4

169

4859

3155

619

3966

1298

472

857

2718

1036

4.0

23

#5

72

2642

2815

311

3152

1441

307

632

1738

893

3.5

16

#6

150

4252

2281

414

3143

1277

257

807

2209

822

4.7

22

Mean

132

3912

2430

424

3281

1142

386

764

2089

827

6

19

RSD

36%

25%

19%

25%

24%

19%

40%

11%

17%

23%

87%

18%

Exposed fish#(ng cm-2)

#1

3.3

129

148

17

 

323

70

 

48

33

89

73

 

1.2

0.9

#2

14.2

394

214

35

255

93

32

68

160

61

0.37

1.5

#3

11.6

310

195

30

220

85

28

56

150

55

0.35

1.0

#4

9.6

275

213

35

268

88

32

49

154

70

0.27

1.3

#5

6.4

233

258

27

289

132

28

56

154

82

0.32

1.4

#6

11.0

312

171

30

236

96

19

59

162

62

0.35

1.6

Mean

9.4

276

200

29

265

94

31

53

145

67

0.5

1.3

RSD

42%

32%

19%

23%

14%

22%

30%

22%

19%

15%

76%

21%

#Since the test chemicals were exposed in two mixtures, each fish # shows the results for 2 individuals, 6 test chemicals in each

Italics = <LOQ

Table: Concentrations (ng cm-2) of test chemicals in mucus from control fish and exposed fish.

 

P9

P12

P13

P16

 

S12

S16

 

T9

T10

T13

T14

 

Q10

Q14

Control fish#

#1

0.19

0.31

0.07

0.03

 

0.09

0.01

 

0.08

0.02

0.15

 

 

0.01

0.09

#2

0.05

0.04

0.04

0.00

 

0.04

0.00

 

0.05

0.01

0.01

 

 

0.00

0.01

#3

0.02

0.03

0.03

0.02

0.04

0.00

0.04

0.01

0.01

0.01

0.01

0.05

LOQ

1.01

1.69

0.27

0.16

0.38

0.06

0.25

0.07

0.89

 

0.06

0.45

Exposed fish#

#1

18

168

125

9

 

106

33

 

19

23

57

39

 

0.21

1.1

#2

22

186

103

10

93

30

17

25

59

31

0.26

1.6

#3

15

149

89

10

88

23

15

18

52

25

0.21

1.5

#4

24

195

112

11

104

36

18

28

63

35

0.22

2.1

#5

26

223

100

14

92

29

12

31

70

30

0.21

1.8

#6

21

216

79

14

74

23

9

25

70

24

0.15

1.4

Mean

21

190

101

11

93

29

15

25

62

31

0.21

1.6

RSD

19%

15%

16%

16%

13%

18%

25%

18%

12%

18%

16%

20%

#Since the test chemicals were exposed in two mixtures, each fish # shows the results for 2 individuals, 6 test chemicals in each

Table: Mass of the fish and tissues sampled (g) 

Fish #

Whole fish

Viscera

(without liver)

Liver

Blood#

Gills

(one side)

MIX 1:1

112

10.94

0.99

0.9

1.98

MIX 1:2

143

15.64

1.98

0.9

2.57

MIX 1:3

153

12.78

1.6

1

2.85

MIX 1:4

143.2

16.4

2.66

0.7

2.98

MIX 1:5

113.6

7.64

0.99

0.4

2.3

MIX 1:6

98

5.53

0.96

1.2

1.5

MIX 2:1

120.7

9.4

0.95

1.4

1.91

MIX 2:2

152.2

13.5

1.4

1.8

2.24

MIX 2:3

165.2

15.5

2.0

2.0

 

MIX 2:4

130.1

8.52

0.96

1.6

2.28

MIX 2:5

145.9

14.14

1.39

1.2

2.33

MIX 2:6

139.3

11.64

1.16

1.9

2.04

#Values given in mL.

Table: Contribution of each tissue to the fish’s body burden of the test chemical.

 

P9

P12

P13

P16

 

S12

S16

 

T9

T10

T13

T14

 

Q10

Q14

Blood

Fish #1

0.016

0.014

0.013

0.013

 

0.021

0.010

 

0.045

0.050

0.038

0.024

 

0.007

0.003

Fish #2

0.017

0.015

0.014

0.011

0.022

0.011

0.059

0.038

0.031

0.027

0.006

0.003

Fish #3

0.021

0.017

0.016

0.012

0.023

0.012

0.075

0.046

0.038

0.030

0.011

0.003

Fish #4

0.010

0.011

0.013

0.010

0.019

0.010

0.046

0.023

0.023

0.028

0.023

0.002

Fish #5

0.012

0.010

0.013

0.008

0.022

0.011

0.056

0.032

0.025

0.024

0.011

0.002

Fish #6

0.018

0.018

0.013

0.016

0.021

0.012

0.062

0.053

0.042

0.025

0.029

0.003

Mean

0.016

0.014

0.014

0.012

 

0.021

0.011

 

0.057

0.041

0.033

0.026

 

0.015

0.003

RSD

25%

23%

10%

21%

6%

9%

20%

29%

23%

8%

64%

22%

Other(all tissue apart from blood, liver, gills and skin; assumed to have the same concentration as muscle)

Fish #1

0.08

0.15

0.17

0.14

 

0.33

0.17

 

0.35

0.54

0.36

0.22

 

0.16

0.09

Fish #2

0.14

0.16

0.17

0.13

0.30

0.16

0.39

0.33

0.29

0.21

0.72

0.08

Fish #3

0.14

0.16

0.24

0.15

0.38

0.20

0.43

0.35

0.31

0.24

0.43

0.07

Fish #4

0.12

0.16

0.17

0.18

0.33

0.12

0.38

0.29

0.32

0.21

0.25

0.06

Fish #5

0.12

0.20

0.17

0.11

0.30

0.14

0.35

0.38

0.31

0.20

0.40

0.05

Fish #6

0.10

0.12

0.17

0.11

0.31

0.18

0.33

0.37

0.29

0.24

0.24

0.05

Mean

0.12

0.16

0.18

0.13

0.32

0.16

0.37

0.38

0.31

0.22

0.37

0.07

RSD

21%

16%

16%

18%

9%

17%

10%

22%

8%

8%

54%

20%

Liver

Fish #1

0.09

0.16

0.24

0.11

 

0.18

0.14

 

0.08

0.07

0.05

0.06

 

0.02

0.05

Fish #2

0.20

0.22

0.23

0.14

0.16

0.12

0.08

0.17

0.09

0.06

0.02

0.06

Fish #3

0.17

0.20

0.16

0.12

0.15

0.10

0.11

0.21

0.11

0.06

0.04

0.05

Fish #4

0.19

0.24

0.15

0.15

0.12

0.08

0.04

0.37

0.21

0.04

0.05

0.06

Fish #5

0.08

0.12

0.23

0.08

0.21

0.12

0.18

0.12

0.07

0.08

0.03

0.04

Fish #6

0.08

0.16

0.27

0.11

0.22

0.13

0.26

0.11

0.06

0.10

0.04

0.05

Mean

0.14

0.19

0.21

0.12

0.17

0.12

0.13

0.18

0.10

0.07

0.03

0.05

RSD

43%

25%

21%

24%

23%

19%

62%

61%

59%

31%

29%

20%

Italics means that the tissue contribution was based mainly on measurements <LOQ


Table (continued): Contribution of each tissue to the fish’s body burden of the test chemical.

 

P9

P12

P13

P16

 

S12

S16

 

T9

T10

T13

T14

 

Q10

Q14

Gills

Fish #1

0.36

0.34

0.27

0.62

 

0.17

0.51

 

0.11

0.14

0.28

0.32

 

0.17

0.75

Fish #2

0.26

0.24

0.27

0.57

0.22

0.51

0.14

0.14

0.26

0.35

0.10

0.67

Fish #3

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

Fish #4

0.28

0.26

0.35

0.50

0.24

0.62

0.15

0.12

0.22

0.40

0.35

0.68

Fish #5

0.35

0.32

0.28

0.65

0.19

0.52

0.14

0.16

0.30

0.34

0.26

0.73

Fish #6

0.26

0.26

0.26

0.58

0.19

0.49

0.11

0.13

0.24

0.30

0.30

0.67

Mean

0.30

0.28

0.28

0.58

 

0.20

0.53

 

0.13

0.14

0.26

0.34

 

0.23

0.70

RSD

17%

16%

13%

10%

15%

10%

15%

10%

13%

11%

43%

5%

Skin

Fish #1

0.07

0.14

0.17

0.08

 

0.23

0.12

 

0.30

0.12

0.17

0.24

 

0.55

0.05

Fish #2

0.15

0.25

0.21

0.12

0.21

0.15

0.22

0.23

0.24

0.24

0.09

0.09

Fish #3

0.16

0.23

0.20

0.10

0.18

0.13

0.17

0.19

0.22

0.22

0.18

0.07

Fish #4

0.11

0.19

0.21

0.12

0.21

0.12

0.24

0.12

0.17

0.21

0.18

0.08

Fish #5

0.09

0.18

0.23

0.10

0.21

0.17

0.19

0.20

0.21

0.26

0.18

0.08

Fish #6

0.19

0.26

0.20

0.13

0.20

0.16

0.17

0.24

0.25

0.23

0.27

0.12

Mean

0.13

0.21

0.20

0.11

0.21

0.14

0.21

0.18

0.21

0.24

0.24

0.08

RSD

35%

22%

10%

15%

9%

15%

24%

28%

18%

7%

66%

29%

Mucus

Fish #1

0.38

0.19

0.14

0.04

 

0.08

0.06

 

0.12

0.08

0.11

0.13

 

0.09

0.06

Fish #2

0.23

0.12

0.10

0.03

0.08

0.05

0.12

0.09

0.09

0.12

0.07

0.10

Fish #3

0.20

0.11

0.09

0.04

0.07

0.03

0.09

0.06

0.07

0.10

0.11

0.11

Fish #4

0.28

0.14

0.11

0.04

0.08

0.05

0.14

0.07

0.07

0.11

0.15

0.12

Fish #5

0.36

0.17

0.09

0.05

0.07

0.04

0.08

0.11

0.09

0.10

0.12

0.10

Fish #6

0.35

0.18

0.09

0.06

0.06

0.04

0.08

0.10

0.11

0.09

0.12

0.11

Mean

0.30

0.15

0.10

0.04

0.07

0.04

0.10

0.08

0.09

0.11

0.11

0.10

RSD

25%

22%

19%

21%

10%

19%

23%

22%

19%

14%

26%

20%

Italics means that the tissue contribution was based mainly on measurements <LOQ


Table: Mucus-water distribution coefficients (DMuc-W, mL cm-2), gill-water distribution coefficients (DG-W, mLcm-2) and apparent bioconcentration factor (BCFapp, L kg-1wet weight). The relative standard deviation (%) is shown in brackets.

 

P9

P12

P13

P16

 

S12

S16

 

T9

T10

T13

T14

 

Q10

Q14

 

DMuc-W

0.47

(19)

16.3

(15)

25

(16)

#

 

5.4

(13)

27

(18)

 

0.29

(25)

1.28

(18)

22

(12)

51

(18)

 

0.0035

(16)

1.40

(20)

DG-W

0.44

(20)

28

(13)

62

(20)

#

13.3

(15)

300

(19)

0.30

(17)

1.95

(10)

58

(17)

150

(21)

0.0064

(15)

9.5

(17)

BCFapp

3.3

230

490

#

150

1260

5.4

32

510

980

0.1

31

# Could not be determined because there were no data for concentrations in water

 

Table: Estimated volume of distribution Vd (L kg-1) of test chemicals (only for resides that had clearly been taken up systemically, i.e., in muscle, liver and blood)§

 

P9

P12

P13

P16

 

S12

S16

 

T9

T10

T13

T14

 

Q10

Q14

Exposed fish#

#1

0.59

1.13

1.59

0.99

 

1.22

1.52

 

0.53

0.65

0.58

0.63

 

-

-

#2

1.06

1.31

1.44

1.20

1.12

1.32

0.45

0.70

0.64

0.54

-

-

#3

0.80

1.07

1.28

1.10

1.17

1.27

0.41

0.65

0.59

0.55

-

-

#4

1.60

1.87

1.33

1.63

1.19

1.06

0.51

1.48

1.18

0.50

-

-

#5

0.83

1.56

1.54

1.15

1.21

1.25

0.52

0.83

0.80

0.61

-

-

#6

0.56

0.79

1.77

0.73

1.28

1.28

0.52

0.49

0.47

0.72

-

-

Mean

0.91

1.29

1.49

1.13

1.20

1.28

0.49

0.80

0.71

0.59

-

-

RSD

43%

30%

12%

26%

5%

12%

10%

44%

36%

14%

-

-

§It was assumed that all tissues except liver, gills, skin and blood contained the same concentration as muscle.

#Since the test chemicals were exposed in two mixtures, each fish # shows the results for 2 individuals, 6 test chemicals in each

Conclusions:
Under the study conditions, the BCFapp for the two quaternary substances: TMAB C10 and TMAC C14 were determined to be 0.1 and 31 L/kg wet weight, respectively, indicating a low systemic uptake or bioaccumulation potential.
Executive summary:

A study was conducted to determine the tissue distribution of two cationic surfactants mixtures in Rainbow Trout (Oncorhynchus mykiss) following exposure via water for seven days and analysis of different fish tissues. The test chemicals were grouped into two mixtures of six containing 10 alkyl amines and 2 quaternary alkylammonium surfactants: TMAB C10 (as part of MIX 2) and TMAC C14 (as part of MIX 1). Studying chemical mixtures has the advantage that differences in behavior between chemicals are not obscured by biological variability or experimental variables. Bioconcentration studies with mixtures have been shown to provide similar results to studies with single chemicals.


The experiments were conducted in 300 L fiberglass aquaria with a water renewal rate of 1.3 L min−1 (MIX 1) and 1.45 L min−1 (MIX 2). A solution of the test chemical mixture in methanol was infused continuously (3.5 and 3.8 μL min−1 for MIX 1 and MIX 2, respectively) into the water inflow using a syringe pump. The intended concentrations of TMAB C10 and TMAC C14 were 59 and 1.3 μg/L (measured). The water temperature was 10 °C and the pH 7.5. The water hardness was estimated to be 1.1 mM Ca2+. For each mixture, the syringe pump was started in an aquarium containing no fish. After 16 h, to allow the concentrations to stabilize, 12 rainbow trout were added. After 7 d of exposure, the fish in the exposure aquaria as well as several unexposed (control) fish were sacrificed followed by blood collection.The surface of the fish posterior of the gills was rinsed with 100% methanol to remove source substance residues adsorbed to the outer surface of the skin and absorbed in the skin mucus.The fish were then dissected and the liver, the kidney, the gills, and the remaining contents of the abdominal cavity were taken and weighed. Skin and muscle samples were prepared from the upper dorsal region on semi-frozen fish after the methanol rinse had removed the mucus. For 6 fish from each aquarium and 3 control fish, samples of muscle, skin, liver, and gills were homogenized in a bullet blender (muscle and liver) or in a cryo-mill (skin and gill). A sub-sample of 0.5−1.2 g of the homogenate was extracted twice in methanol, employing centrifugation at 4000 rpm for phase separation. Isotope labeled standards of TMAB C10 and TMAC C14 were added to a portion of the extract corresponding to 12−75 mg of the sample. Whole blood was analyzed rather than plasma because of the small quantity of sample available and the anticipated low concentrations.


The test chemical concentrations generally increased in the order muscle <blood < skin < gills < liver. Because the mass of extracted mucus was not determined, the concentrations in mucus were normalized to the estimated fish’s total surface area excluding the head, which was not rinsed. The concentration in mucus was on average 3.9 (range 0.9−11.6) times lower than the surface area-normalized concentration in gills. To calculate the quantity of the test chemical in the different tissues, the amount of each tissue in the fish was estimated and multiplied by the concentration in that tissue. The test chemical quantities in the different tissues were then summed to give the body burden in each fish. The apparent BCFs (BCFapp) values at the end of the 7-day exposure were calculated by dividing the surfactant body burden (blood, muscles, liver, gills, skin, mucus) by the fish mass, and dividing this by the average measured concentration in water samples taken during the exposure phase. Under the study conditions, the BCFapp for the two quaternary substances TMAB C10 and TMAC C14 were determined to be 0.1 and 31 L/kg wet weight, respectively. Mucus, skin, gills, liver, and muscle each contributed at least 10% of body burden for the majority of the test chemicals. In contrast to the analogue alkylamine bases, the permanently charged quaternary ammonium compounds accumulated mostly in the gills and were nearly absent in internal tissues, indicating that systemic uptake of the charged form of cationic surfactants is very slow (Kierkegaard, 2020). Based on the results of the source study, a similar low bioaccumulation potential is expected for the target substance.

Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Information on the category justification can be found in the Quaternary ammonium salts (QAS) category and section 13.2 of IUCLID.
Qualifier:
according to guideline
Guideline:
EPA OPP 165-4 (Laboratory Studies of Pesticide Accumulation in Fish)
Deviations:
no
GLP compliance:
yes
Radiolabelling:
yes
Details on sampling:
- A stock solution of 2.4 mg/mL was formulated by mixing radiolabeled test substance with unlabeled test substance and diluting with water.
- Following dilution with unlabeled test substance, the percent radiolabeled test substance in the mixture was 0.337 or 0.00337 as a decimal. On a combined test substance basis (both labeled and unlabeled) the calculated specific activity was 227013 dpmug x 0.00337 = 765.03 dpm/ug
Test organisms (species):
Lepomis macrochirus
Details on test organisms:
Test organism:
- Common name: Bluegill
- Strain: SLS Lot 88A9
- Source: Froma culture maintained at Springborn Life Sciences, Inc.
- Length at study initiation (lenght definition, mean, range and SD): 65 mm
- Weight at study initiation (mean and range, SD): 3.6g
- Health status: Normal and healthy
- Frequency: Daily

Acclimation:
- Acclimation period: 14d prior to test initiation
- Acclimation conditions (same as test or not): Yes
- Type and amount of food: Dry pelleted food, ad libitum, daily throughout the 14d period, except 24h before testing.
Route of exposure:
aqueous
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
ca. 35 d
Total depuration duration:
ca. 21 d
Hardness:
22-32 mg/L CaCO3
Test temperature:
16-21°C
pH:
6.7-7.6
Dissolved oxygen:
6.2 -10.1 mg/L (64-94% of saturation)
Details on test conditions:
Test system
- Test vessel: 76 x 40 x 30 cm aquaria
- Type (delete if not applicable): Open
- Material, size, headspace, fill volume: 75L
- Renewal rate of test solution (frequency/flow rate): 4-4.7 tank volume replacements/d; 2L water to each aquaria at an average rate of 375 times/d
- No. of organisms per vessel: 190
- Biomass loading rate: 0.91 g/L/day

Test medium / Water parameters:
- Source/preparation of dilution water: Dilution water used for this test was from the same source as the water which flowed into the fish holding tank and was characterised weekly.
- Alkalinity: 21-24 mg/L CaCO3
- Conductance: 100-130 umhos/cm

Other test conditions:
- Photoperiod: 16h light/ 8h dark

Sampling period:
- To monitor initial concentration of 14C residues in water: 5 mL of samples were collected on Days 4, 3, 2, and 1 before introduction of fish
- To monitor concentration of 14C residues in water during the test: 5 mL of samples were collected on Days 1, 3, 7, 8, 9, 10, 14, 21, 23, 28 and 35 of exposure
- To quantify the accumulation and elimination of 14C residues in the edible, non-edible tissue of fish: 5 fish were collected, eviscerated and filleted on Day 1, 3, 7, 10, 14, 21, 28 and 35 of exposure.
- To quantify background 14C residues of test substance: 5 control fish were also collected, eviscerated and filleted on Day 35 of exposure and Day 21 of depuration.
- To estimate the half-life of the accumulated 14C residue present in the fish during depuration period: Water and tissue samples were collected from the tankon Day 1, 3, 7, 10, 14, 16 and 21 of depuration. Five fish were collected at each interval for analysis.

Others:
- Steady state was determined by measuring 14C residue conc. for 3 consecutive sampling intervals.
- After 35d of exposure, 35 of remaining fish in the treatment aquarium were transferred to a clean aquarium into which untreated dilution water was introduced at a rate equal to flow rate during exposure.
- Triplicate edible fish tissue samples from Day 35 of exposure were prepared for a hexane and methanol extraction procedure.
- To estimate the amount of 14C residues bound to the external surface of the skin of fish: 10 fish were removed after 35 d of exposure, eviscerated, filleted and the muscle portions were scraped off the skin. The skin portions were subsequently combustible to determine the residue conc. of the test substance.
Nominal and measured concentrations:
Nominal concentration: 0.05 mg/L
Measured concentration: 0.076 mg/L
Details on estimation of bioconcentration:
BCF factors for edible, non-edible and whole body fish tissue were determined by dividing the mean measured equilibrium 14C tissue concentration for each tissue type by the mean measured water concentration for the entire exposure period. For comparison, an additonal method of calculating BCF factors wher the ratio of the uptake constant (Ku) to the depuration constant (Kd) was utilized. i.e.,
BCF = Ku/Kd

Key result
Type:
BCF
Value:
79 dimensionless
Basis:
whole body w.w.
Calculation basis:
other: mean
Remarks on result:
other: predicted BCF (calculated using uptake and depuration constants) = 110
Remarks:
conc. in environment / dose: 0.076 mg/L
Key result
Type:
BCF
Value:
160 dimensionless
Basis:
non-edible fraction
Calculation basis:
other: mean
Remarks on result:
other: predicted BCF = 190
Remarks:
conc. in environment / dose: 0.076 mg/L
Key result
Type:
BCF
Value:
33 dimensionless
Basis:
edible fraction
Calculation basis:
other: mean
Remarks on result:
other: predicted BCF = 50
Remarks:
conc. in environment / dose: 0.076 mg/L
Key result
Elimination:
yes
Parameter:
DT50
Depuration time (DT):
21 d
Details on results:
- Mortality of test organisms: two treated fish (in a population of 190) died
- Behavioural abnormalities: None

Exposure phase:
- The mean 14C residues measured in the edible tissue reached steady state on Day 14 and remained relatively constant throughout the remainder of the exposure (mean range 2.10-3.36 mg/kg).
- Mean steady state conc. in edible tissue and mean conc. in the water during the period 0-35d: 2.54±0.67 mg/kg and 0.076±0.024 mg/L. The mean steady state BCF factor in the edible tissue during the 35d of exposure was determined to be 33X.
- The mean 14C residues measured in the non-edible tissue reached steady state on Day 14 and ranged from 11-13 mg/kg during the remainder of the 35-d exposure.
- Mean steady state in non-edible tissue and mean conc. in the water during the period 0-35 d: 12±2.3 mg/kg and 0.076±0.024 mg/L. The mean steady state BCF factor in the non-edible tissue during the 35d of exposure was determined to be 160X.
- The mean steady state conc. of 14C residue conc. in whole fish and mean conc. in the water during the period 0.35 d: 6±1.5 mg/kg and 0.076±0.024 mg/L.. The mean steady state BCF factor in the whole body of fish during the 35d of exposure was determined to be 79X.
- The residues in control were below the detectable level; small residues seen were due to contamination.
- Analytical results of the polar (methanol) solvent and non-polar (hexane) solvent extractions of edible tissue samples revealed 11% of the 14C residues accumulated in the exposure were extractable with methanol, 2.4% with hexane and 75% were not extractable with either solvent.

Depuration phase:
- Analyses of the tissues during the depuration phase indicated that in each case there was a continuous elimination of 14C residues from the respective tissues over the course of the 21d depuration period.
- Concentration of 14C residues present in the water of the depuration aquarium remained <= 0.014 mg/L, the limit of radiometric detection throughout the 21d depuration period.
- Half-life for non-edible tissue was: Between Days 14 and 21. By Day 21, the fish had eliminated 29%, 60% and 44% of the 14C residues that had been present on the last day of exposure on each of the tissues (i.e., edible, non-edible and whole body tissues respectively).
- Half-life based on model calculations (using 1st order kinetics) were: 16, 10 and 12d for edible, non-edible and whole body tissues respectively.
- The conc. of the residual test substance on the skin was observed to be higher than those found in the total edible tissue (skin and muscle combined) indicating significant binding of the test substance to skin and scales of fish.


Given the known strong affinity for the cationic compounds like the test substance to bind to essentially all surfaces, a logical explanation for the observed slow depuration is that the 14C residues are strongly bound to surfaces exposed to the treated water, e.g., gills, skin and intestine. The phenomenon has been clearly demonstrated with other quarternary ammonium compounds.

Validity criteria fulfilled:
not specified
Conclusions:
Under the study conditions, the whole body BCF of the source substance was determined to be 79, indicating low potential to bioaccumulate.
Executive summary:

A study was conducted to determine the aquatic bioaccumulation of the source substance, C12-16 ADBAC (30.64% active; 98.9% radiolabeled purity) in Lepomis macrochirus (bluegill fish) under flow-through conditions, according to EPA OPP 165-4, in compliance with GLP. The blue gill fish were continuously exposed to a nominal concentration of 0.050 mg/L of the source substance (equivalent to a measured concentration of 0.076 mg/L) in well water for 35 days, followed by transfer of 35 fish into flowing uncontaminated water for a 21-d depuration period. Sampling was carried out on Days 0, 1, 3, 7, 9, 10, 14, 21, 23, 28 and 35 for the exposure period and Days 1, 3, 7, 10, 14 and 21 for the depuration period. Water samples were collected on Day 8 of the exposure period and Day 16 of the depuration for analytic determination of the source substance concentration. Radiometric analyses of the water and selected fish tissues revealed that the mean steady state bioconcentration factor (BCF) in the edible, non-edible and whole-body fish tissue during the 35 days of exposure to be 33, 160 and 79 L/kg. The half-life for non-edible tissue was attained between Days 14 and 21, while it could not be reached for the edible and whole-body fish tissues by the end of 21-d depuration period. By Day 21 of the depuration period, the 14C residues present on the last day of exposure in the edible, non-edible and whole-body fish tissues had been eliminated by 29, 60 and 44% respectively. Analysis of skin tissue after 35 d of exposure showed residue levels somewhat higher than those observed for edible tissue at the same sampling period, indicating that there is likely significant binding of 14C ADBAC to the skins and scales of exposed bluegill, as expected behaviour of cationic surfactants. Under the conditions of the study, the whole body BCF of the source substance was determined to be 79, indicating low potential to bioaccumulate (Fackler, 1989). Based on the results of the source study, a similar low bioaccumulation potential is expected for the target substance.​

Description of key information

The results of the study with a structurally similar substance, supported with the estimated BCF value for the registered substance together with its ionic nature indicates a low bioaccumulation and biomagnification potential. The higher experimental BCF value of 79 L/kg wt-wt from the study with C12-16 ADBAC and the growth corrected kinetic biomagnification factor (BMFkg) value of 0.0463 based on read across to TMAC C18, has been considered further for hazard/risk assessment. 

Key value for chemical safety assessment

BCF (aquatic species):
79 L/kg ww
BMF in fish (dimensionless):
0.046

Additional information

Study 1:A study was conducted to determine the aquatic bioaccumulation of the source substance, C12-16 ADBAC (30.64% active; 98.9% radiolabeled purity) inLepomis macrochirus(bluegill fish) under flow-through conditions, according to EPA OPP 165-4, in compliance with GLP. The blue gill fish were continuously exposed to a nominal concentration of 0.050 mg/L of the source substance (equivalent to a measured concentration of 0.076 mg/L) in well water for 35 days, followed by transfer of 35 fish into flowing uncontaminated water for a 21-d depuration period. Sampling was carried out on Days 0, 1, 3, 7, 9, 10, 14, 21, 23, 28 and 35 for the exposure period and Days 1, 3, 7, 10, 14 and 21 for the depuration period. Water samples were collected on Day 8 of the exposure period and Day 16 of the depuration for analytic determination of the source substance concentration. Radiometric analyses of the water and selected fish tissues revealed that the mean steady state bioconcentration factor (BCF) in the edible, non-edible and whole-body fish tissue during the 35 days of exposure to be 33, 160 and 79 L/kg. The half-life for non-edible tissue was attained between Days 14 and 21, while it could not be reached for the edible and whole-body fish tissues by the end of 21-d depuration period. By Day 21 of the depuration period, the 14C residues present on the last day of exposure in the edible, non-edible and whole-body fish tissues had been eliminated by 29, 60 and 44% respectively. Analysis of skin tissue after 35 d of exposure showed residue levels somewhat higher than those observed for edible tissue at the same sampling period, indicating that there is likely significant binding of 14C-ADBAC to the skins and scales of exposed bluegill, as expected behaviour of cationic surfactants. Under the conditions of the study, the whole body BCF of the source substance was determined to be 79, indicating low potential to bioaccumulate (Fackler, 1989).  


Study 2: A study was conducted to determine the biomagnification (BMF) potential of the source substance, TMAC C18 (purity 95%), following the principles of OECD TG 305. For the main study rainbow trout (Oncorhynchus mykiss) with an average weight of 5.42 g were fed test diets enriched with source substance (23.6 mg/kg read across the substance in feed. The resulting treatment and one control group (each 40 animals) were tested simultaneously. The uptake phase of 14 days was followed by a depuration phase lasting 14 days. All animals were fed the non-spiked feed during the depuration phase. The concentrations of the source substance in fish samples were determined by chemical analysis and all tissue concentrations were calculated based on a wet weight basis. Chemical analysis of the source substance was performed by liquid chromatography with coupled mass spectrometry (LC-MS/MS). In the main study five animals of each group were sampled randomized on Day 7 and Day 14 of the uptake phase and after 10 h, 24 h, 2 days, 3 days, 7 days and 14 days of depuration. Biomagnification factor (BMF) and distribution factor were calculated based on the tissue concentrations measured at the end of the uptake phase. No mortality or abnormal behaviour of the test animals was observed during the main study. The experimental diets were accepted by the test animals and showed a decent digestibility as confirmed by the texture and appearance of the feces. One fish was euthanized at Day 25 due to injuries. The specific growth rates of the animals ranged from 1.95 to 2.71 %/d over the entire experiment. During the study, the feed conversion ratio (FCR) was 0.69 to 0.95. Fish were measured and weighed at the beginning of the experiment as well as at respective sampling time points to monitor growth and associated growth-dilution effects during the feeding study. Growth rate constants were determined separately for the uptake and depuration phases, for the treatments and the control group, using the ln-transformed weights of the fish. A subsequent parallel line analysis (PLA, as suggested by the OECD Guideline) resulted in no statistical differences between the uptake and the depuration phase among the treated groups with the source substance. No statistically significant difference was detected with regard to the growth of the treated groups. Hence it was deduced that neither adverse nor toxic effects were caused by the enriched diets. As steady state seemed to be reached after 14 days of exposure, steady state biomagnification factors (BMFss) could be calculated as 0.02709 g/g, which showed that source substance did not biomagnify after dietary exposure. In general, the GIT and the liver showed the highest values for the BMFk and BMFkg. The kinetic BMF (BMFk) and growth-corrected biomagnification factor (BMFkg) were calculated for the source substance to be 0.0404 and 0.0463, respectively. Overall, it was concluded from the screening that ionization lowers the tendency of a chemical to bioaccumulate, compared to non-ionized chemicals. Aside from the well-known lipophobicity of ionized groups, fast depuration seems to be a major reason for the observed low biomagnification of ionic compounds, in particular anions. Fast depuration may happen due to rapid metabolism or conjugation of charged compounds, and future studies should test this hypothesis. Under the study conditions, the source substance BMFss, BMFk and BMFkg values on whole body wet weight basis in rainbow trout were determined to be 0.02709, 0.0404 and 0.0463 g/g, respectively, suggesting low biomagnification potential (Schlechtriem, 2021). Based on the results of the read across study, a similar low biomagnification potential is expected for the target substance. 


Study 3:A study was conducted to determine the tissue distribution of two cationic surfactants mixtures in Rainbow Trout (Oncorhynchus mykiss) following exposure via water for seven days and analysis of different fish tissues. The test chemicals were grouped into two mixtures of six containing 10 alkyl amines and 2 quaternary alkylammonium surfactants: TMAB C10 (as part of MIX 2) and TMAC C14 (as part of MIX 1). Studying chemical mixtures has the advantage that differences in behavior between chemicals are not obscured by biological variability or experimental variables. Bioconcentration studies with mixtures have been shown to provide similar results to studies with single chemicals. The experiments were conducted in 300 L fiberglass aquaria with a water renewal rate of 1.3 L min−1 (MIX 1) and 1.45 L min−1 (MIX 2). A solution of the test chemical mixture in methanol was infused continuously (3.5 and 3.8 μL min−1 for MIX 1 and MIX 2, respectively) into the water inflow using a syringe pump. The intended concentrations of TMAB C10 and TMAC C14 were 59 and 1.3 μg/L (measured). The water temperature was 10 °C and the pH 7.5. The water hardness was estimated to be 1.1 mM Ca2+. For each mixture, the syringe pump was started in an aquarium containing no fish. After 16 h, to allow the concentrations to stabilize, 12 rainbow trout were added. After 7 d of exposure, the fish in the exposure aquaria as well as several unexposed (control) fish were sacrificed followed by blood collection. The surface of the fish posterior of the gills was rinsed with 100% methanol to remove source substance residues adsorbed to the outer surface of the skin and absorbed in the skin mucus. The fish were then dissected and the liver, the kidney, the gills, and the remaining contents of the abdominal cavity were taken and weighed. Skin and muscle samples were prepared from the upper dorsal region on semi-frozen fish after the methanol rinse had removed the mucus. For 6 fish from each aquarium and 3 control fish, samples of muscle, skin, liver, and gills were homogenized in a bullet blender (muscle and liver) or in a cryo-mill (skin and gill). A sub-sample of 0.5−1.2 g of the homogenate was extracted twice in methanol, employing centrifugation at 4000 rpm for phase separation. Isotope labeled standards of TMAB C10 and TMAC C14 were added to a portion of the extract corresponding to 12−75 mg of the sample. Whole blood was analyzed rather than plasma because of the small quantity of sample available and the anticipated low concentrations. The test chemical concentrations generally increased in the order muscle <blood < skin < gills < liver. Because the mass of extracted mucus was not determined, the concentrations in mucus were normalized to the estimated fish’s total surface area excluding the head, which was not rinsed. The concentration in mucus was on average 3.9 (range 0.9−11.6) times lower than the surface area-normalized concentration in gills. To calculate the quantity of the test chemical in the different tissues, the amount of each tissue in the fish was estimated and multiplied by the concentration in that tissue. The test chemical quantities in the different tissues were then summed to give the body burden in each fish. The apparent BCFs (BCFapp) values at the end of the 7-day exposure were calculated by dividing the surfactant body burden (blood, muscles, liver, gills, skin, mucus) by the fish mass, and dividing this by the average measured concentration in water samples taken during the exposure phase. Under the study conditions, the BCFapp for the two quaternary substances TMAB C10 and TMAC C14 were determined to be 0.1 and 31 L/kg ww, respectively. Mucus, skin, gills, liver, and muscle each contributed at least 10% of body burden for the majority of the test chemicals. In contrast to the analogue alkylamine bases, the permanently charged quaternary ammonium compounds accumulated mostly in the gills and were nearly absent in internal tissues, indicating that systemic uptake of the charged form of cationic surfactants is very slow (Kierkegaard, 2020).  


Study 4:The Bioconcentration factor (BCF) value of target substance, TMAC T was predicted using regression-based and Arnot-Gobas BAF-BCF models of BCFBAF v3.02 program (EPI SuiteTMv4.11). The Arnot-Gobas method, takes into account mitigating factors, like growth dilution and metabolic biotransformations, therefore the BCF values using this method is considered to be more realistic or accurate. Therefore, except for ionic, pigments and dyes, perfluorinated substances, for which it is not recommended (as of now), the Arnot-Gobas method is used preferentially used for BCF predictions. Considering that the target substance is an UVCB containing majorly ionic (e.g., (e.g., the quaternary ammonium salts) and few non-ionic constituents (e.g., amines), the BCF values were predicted using regression-based and Arnot-Gobas BAF-BCF models respectively and using SMILES codes as the input parameter. The BCF values for the constituents ranged from 3.16 to 162.4 L/kg ww (log BCF: 0.50 to 2.21), indicating a low bioaccumulation potential. On comparing with domain descriptors, all constituents were found to meet the MW, log Kow and/or maximum number of correction factor instances domain criteria as defined in the BCFBAF user guide of EPISuite. Further, given that the major constituents are structurally very similar and vary only in the carbon chain length, a weighted average value, which takes into account the percentage of the constituent in the substance, has been considered to dampen the errors in predictions (if any). Therefore, the weighted average BCF value was calculated as 70.3 L/Kg ww (Log BCF = 1.85). Overall, considering either the individual BCF predictions for the constituents or the weighted average values, the target substance is expected to have a low bioaccumulation potential. However, taking into consideration the model’s training set and validation set statistics and the fact that the training set only contains 61 ionic compounds, the BCF predictions for the individual constituents are considered to be reliable with moderate confidence. 


This is further supported by the no bioaccumulation potential evidence observed in in the two toxicokinetic studies in mammals with the source substance, C12-16 ADBAC (Selim, 1987 and Appelqvist, 2006). 


Also, the biocides assessment reports available from RMS Italy on TMAC C (as Coco TMAC) and C12-16 ADBAC, concluded the substances to show low potential for bioaccumulation, based on the results from the above study (Fackler, 1989) and an additional read across to DDAC for the TMAC C assessment ((ECHA biocides assessment report, 2015, 2016). The report concluded the following in the TMAC C assessment report:“Coco alkyltrimethylammonium chloride is readily biodegradable, is rapidly excreted and does not accumulate in mammals, and it adsorbs onto the fish surface where its irritating action is expressed (therefore accumulation is more related to the concentration of the administered solution). Based on these properties’ bioaccumulation is not expected to be of concern for ATMAC/TMAC. An experimental BCFwhole body of 81 L/kg was determined in a flow-through test with Lepomis machrochirus and the source substance DDAC (Lonza Cologne GmbH and Akzo Nobel Surface Chemistry AB, same study). A very similar result was obtained for the other quaternary ammonium compound benzyl-C12-16-alkyldimethyl ammonium chloride (C12-16-BKC/ADBAC) in a fish bioconcentration test, which gave a BCFwhole body = 79 L/kg (Akzo Nobel Surface Chemistry AB, access to Lonza Cologne GmbH study). Being both studies equally reliable, the BCFwhole body = 81 L/kg is chosen because related to the lead source substance (DDAC) and it is slightly higher than the C12-16 BKC/ADBAC endpoint.”  


Overall, the results of the read across study, supported with the estimated BCF value for the target substance together with its ionic nature indicates a low bioaccumulation and biomagnification potential. The higher experimental BCF value of 79 L/kg wt-wt from the read across study with C12-16 ADBAC and the growth corrected kinetic biomagnification factor (BMFkg) value of 0.0463 based on read across to TMAC C18, has been considered further for hazard/risk assessment.