Dimethyldioctylammonium chloride

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 May 2000 to 30 March 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD Guideline No. 210

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

A combination of didecyldimethylammonium chloride was used with [14C] labelled didecyldimethylammonium chloride as follows:
Didecyldimethylammonium chloride:
-Trade name - Bardac 22
- Composition - didecyldimethylammonium chloride (51.4%) in propanol and water
- CAS nnumber - 7173-51-5
- Physical appearance - pale yellow liquid
- Batch number - 106 016 9991
- Expiration date - not provided
- Reciept date - 20 April 200

Radiolabelled test substance
- Name - [14C] didecyldimethylammonium chloride
- specific activity - 23.39 mCi/mmol
- radiochemical purity - 99.76%
- Lot Number - 990709
- Date of receipt - 22/07/1999

Analytical monitoring:

yes

Details on sampling:

- Methods: The dosing and stability of the test substance in the aquesous solutions of the flow through test were checked using Liquid Scintillation Counting (LSC) and HPLC.
- Analysis time points: Aliquots of 10ml were sampled at 0, 7, 14, 21, 28 and 34 days for all test substance concentrations for LSC analysis.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

- Eggs were obtained from the Commercial fish hatchery Atlant in Hellevoetsluis, the Netherlands.They were confirmed as blastula statge.
- All eggs were from the same batch.

Test type:

other: Intermittent flow through

Water media type:

other: synthetic DSWL-E medium prepared from ground water.

Limit test:

no

Total exposure duration:

34 d

Hardness:

Hardness of the medium was analysed as 238 mg/L.

Test temperature:

23.4 - 25.2 °C

pH:

7.7 - 8.2 in all the test substance concentrations

Dissolved oxygen:

The dissolved oxygen ranged from 6.7 to 9.3 mg/L. The dissolved oxygen concentration decreased over the duration of the experiment.

Salinity:

Not stated

Conductivity:

Not stated

Nominal and measured concentrations:

- Nominal concentrations - 1, 3.2, 10, 32, 100 and 320 µg/L.
- Ratio of labelled and unlabelled test substance - 15/85% ratio for 1, 3.2, 10 and 32 µg/L, 1.5/98.5% ratio for 100 and 320 µg/L.
- All observations on the test solutions showed they were clear.
- Based on the LSC analysis of the test solutions the average dosed concentration for all test concentrations at each measured time point was 115.2% of the nominal concentrations. The full details of each concentration are included in Table 1. Based on HPLC analysis it was found that didecyldimethylammoniumchloride was 100% present during the test and no degradation products were detectable, see Table 2.
- The measured concentrations based on HPLC were in average from the 6 time points were 1.2 ± 0.2 µ/L, 4 ± 0.6 µ/L, 12 ± 1.3 µ/L, 35.1± 3.2 µ/L, 106 ± 10 µ/L, 334 ± 2 µ/L

Details on test conditions:

Preliminary study
- Semi static range finding test was used to determine appropriate final test concentrations. No other details provided.

Stock solutions
- new stock solutions were prepared weekly and stored in the refridgerator until use.
- dosed volume of stock solution was ca. 0.1ml per litre of diultion water.

Intermittent flow through design
- constructed according to Van Leeuwen et al. See attached figures below.
- Process - Every 51 minutes valve 1a opens and dilutions are supplied from a reservoir tank (1) to a glass splitter (2) consisting of 8 one litre comparments. The 8 compartments are succesively filled until the water level reaches a sensor. The water supply is then cut off and valves (3) under the compartments are activated and opened, causing the water to flow into the mixing bottles. At this same moment the test substance is dosed int to the mixing bottle via a syringe pump (11) and mixed with dilution water by magnetic stirrers (9). The one litre volume of each concentration then flows over into the chamber which has total volume of 2.7 litre. The fish are confined to the test chambers in 4 cylindrical retention chambers with a volume of 272ml each. Each chamber recieves about 250ml per cycle by means of a flow splitting funnel. One litre of each concentration per cycle flows out.
- Summary - every 51 minutes each chamber recieves approximately 92% refreshed exposure solution.

Other test parameters:
- Light regime - 16 hour light, 8 hour dark
- At the start of the test 40 potentially fertilised eggs were placed in each test compartment, which was reduced to 20 fertilised eggs after the first 24 hours of exposure, enabling initiation of the test early on in the developmental process. Immediately after hatching the larvae were fed abundantly with rotifers from mass cultures of Artemi nauplii and rotifers from day 10 days onwards.
- No aeration of test solutions and control media.
- pH and oxygen was measured weekly, temperature was measured continuously in the control vessel and once per week in all test vessels.

Parameters measured
- mortality
measurements of length, dry weight of fish at the end of the exposure period
- hatching of eggs was followed daily until all eggs were hatched, after hatching observations were made 3 times per week. The dead eggs or larvae were counted and removed daily and after hatching 3 times per week. The survivors were also counted and their size and condition (swimming behaviour, malformations, or any other visually observable morphological or behavioural criterion) and were compared with that of the control animals.

Statistics
- Statistical significance for mortality was determined using a binomial test at a 95% signficance level, combining the results of the quadruplicates.
- Statistical significance for growth was determined with the two-tailed Dunnett-test with a 95% and 99% significance level.
- The NOEC for condition was not determined statistically.

Reference substance (positive control):

no

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

320 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Key result

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

32 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

eggs, larvae

Key result

Duration:

34 d

Dose descriptor:

LOEC

Effect conc.:

100 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

eggs/larvae

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

32 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: condition

Duration:

34 d

Dose descriptor:

LOEC

Effect conc.:

100 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: condition

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

32 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

34 d

Dose descriptor:

LOEC

Effect conc.:

100 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Hatching and mortality
- The hatching of eggs was not affected ay any concentrations tested.
- Mortality of eggs was affected for concentrations of greater than 32µ/L.
- The results of the early life stage test are presented in Tables 4 and 5 below and the raw data are attached below also.

Visual observations
- In both the control and all exposure concentrations the surviving larvae swam and fed actively during the exposure period and no malformations were noted during the test. However, some visual observations were noted that larvae at 100 µg/L concentrations were smaller than the controls, however length and weight measurements did not confirm this. Based on these observations and the observed mortality the NOEC and LOEC values for condition are estimated to be 32 and 100 µg/L.

Growth
- There was no significant effect on length at any concentration and no signficant effect on weight on any concentration other than the 100 µg/L. At 100 µg/L there was one big fish larva, which was suggested was possibly oedemic, resulting in weight increase.

Results with reference substance (positive control):

N/A

Table 4. Results of the early life stage test with didecyldimethylammonium chloride

^Parameter (d=days)	^Effect	^Concentration (95% confidence interval) in µg/L
^6d LC50	^Mortality	^>320
^8d LC50	^Mortality	^268 (236-303)
^10d LC50	^Mortality	^219 (190-251)
^13d LC50	^Mortality	^173 (150-200)
^15d LC50	^Mortality	^153 (133-76)
^17d LC50	^Mortality	^138 (120-157)
^20d LC50	^Mortality	^120 (106-136)
^22d LC50	^Mortality	^112 (99 -125)
^24d LC50	^Mortality	^104 (93-117)
^27d LC50	^Mortality	^95 (85-107)
^29d LC50	^Mortality	^90 (80-102)
^31d LC50	^Mortality	^86 (76-98)
^34d LC50	^Mortality	^81 (70-93)
^34d NOEC	^Mortality	^32
^34d LOEC	^Mortality	^100
^34d NOEC	^Condition	^32
^34d LOEC	^Condition	^100
^34d NOEC	^Growth	^32
^34d LOEC	^Growth	^100

Table 5. Summary results on hatching, mortality and growth of eggs/larvae of Brachydanio rerio exposed to dideclydimethylammonium chloride.

^Concentration of didecyldimethylammonium chloride	^% of eggs hatched after 6d	^% mortality after 34d	^Growth
			^No of fish	^Length (cm)a	^Dry weight (mg)a
^0	^100	^2.5	^78	^1.32 ± 0.17	^1.83 ± 0.99
^1	^100	^8.8	^73	^1.36 ± 0.15	^2.32 ± 0.44
^3.2	^100	^1.2	^79	^1.3 ± 0.14	^1.96 ± 0.18
^10	^100	^5.0	^76	^1.31 ± 0.19	^2.31 ±0.83
^32	^100	^6.2	^75	^1.34 ± 0.15	^2.11 ± 0.27
^100	^100	^66b	^27	^1.3 ± 0.25	^3.49 ± 1.71c
^320	^100	^100	^0	^-	^-

a) mean and standard deviation (dry weight calculated as explained in the text)

b) mortality significantly higher than that of the control animals (binomial test, p=0.05)

c) dry weight signficantly higher than control (two tailed Dunnet test, p=0.05)

Validity criteria fulfilled:

yes

Conclusions:

The NOEC for Danio rerio (previous name: Brachydanio rerio) exposed to didecyldimethylammonium chloride (c10) for 34 days based on mortality, condition and growth was 32 µg/L.

Executive summary:

An early life stage toxicity to fish test using didecyldimethylammonium chloride (C10, CAS number 7173 -51 -5) was carried out according to GLP and to OECD 210 guidelines. The study was run as an intermittent flow through experiment and test solutions analysed to confirm exposure concentrations of 1.2 ± 0.2 µ/L, 4 ± 0.6 µ/L, 12 ± 1.3 µ/L, 35.1± 3.2 µ/L, 106 ± 10 µ/L, 334 ± 2 µ/L. Fish were added to the tanks at the blastula stage and the study was continued for 34 days. The NOEC based on mortality, condition and growth was 32 µg/L. The LC50 at 34 days was calculated at 81 µg/L (70 to 93µg/L).

Reference 2

Endpoint:

fish early-life stage toxicity

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across to K1 study therefore K2 is the maximum Klimisch value.

Justification for type of information:

Read-across approach - see read-across justification in section 13.

Reason / purpose for cross-reference:

read-across source

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Water samples were collected from mid-depth in one test chamber of each treatment and control group two days prior to the start of the test to confirm the operation of the diluter. Water samples also were collected from mid-depth in alternating replicate test chambers at the beginning of the test, at the completion of hatch on Day 5, at approximately weekly intervals during the test, and at the end of the test to determine concentrations of the test substance. At each sampling interval, an additional sample was collected from the test solution flowing from the diluter mixing chamber for each treatment and control group. This sample was collected from the “splitter tube” that delivered solution to the same replicate test chamber as was sampled from mid-depth in the respective test chambers.

Vehicle:

yes

Details on test solutions:

Stock solutions were prepared approximately weekly during the test. All glassware used in preparation of the stock solutions was acid washed in 10 % HCl prior to use. The stock and test concentrations were prepared on the basis of active ingredient (AI) of the test substance. A primary stock solution was prepared in DMF at a nominal concentration of 1670 μg AI/mL.
Proportional dilutions of the primary stock were made in DMF to prepare additional stock solutions at nominal concentrations of 200, 340, 580 and 980 μg AI/mL. The stock solutions were mixed by inversion, and were clear and colorless in appearance. The stock solutions were delivered to the diluter mixing chambers (at a rate of 12.5 μL/minute) where they were mixed with dilution water (at a rate of 125 mL/minute) to achieve the desired nominal test concentrations of 20, 34, 58, 98 and 167 μg AI/L. The solvent control was prepared by injecting DMF into the mixing chamber assigned to the solvent control.
The concentration of DMF in the solvent control and all treatment groups was 0.1 mL/L. The test solutions appeared clear and colorless in the diluter mixing chambers and in the test chambers at test initiation and termination.

Test organisms (species):

Pimephales promelas

Details on test organisms:

The fathead minnow, Pimephales promelas, was selected as the test species for this study. Fathead minnows are one of the preferred fish species to test the toxicity of test substances during the early life-stages of fish. This species was selected for use in the test based upon past use and ease of handling in the laboratory.
The embryos were removed from spawning substrates and examined under a dissecting microscope to select healthy, viable specimens at approximately the same stage of development. Embryos collected for use in the test were from 10 individual spawns and were < 24 hours old when the test was initiated. To initiate the test, groups of 1 or 2 embryos were randomly distributed among preconditioned incubation cups until each cup contained at least 20 embryos. The cups were immersed in a glass beaker containing the appropriate test solution during the distribution procedure. Care was taken to avoid cross-contamination by discarding the pipette used to transfer the embryos after each transfer. Once each cup contained at least 20 embryos, one cup was placed in each treatment and control test chamber.
Newly-hatched larvae were fed live brine shrimp nauplii (Artemia sp.) three times per day during the first seven days post-hatch, and then three times per day on weekdays and twice daily on weekends throughout the remainder of the test. There were no known levels of contaminants reasonably expected to be present in the diet that were considered to interfere with the purpose or conduct of the test. Fish were not fed for approximately 48 hours prior to the termination of the test to allow for clearance of the digestive tracts before weight measurements were made. To ensure that the feeding rate per fish remained constant, rations were adjusted each week to account for losses due to mortality. Excess feed was siphoned from the test chambers periodically during the test.
Biomass loading at the end of the test, based on the mean wet weight of the negative control group, was 0.037 g of fish per liter of test solution that passed through the test chamber during a 24-hour period.
Instantaneous loading (the total wet weight of fish per liter of water in the tank), based on the mean wet weight of the negative control group at the end of the test, was 0.24 g fish/L.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

33 d

Remarks on exposure duration:

33 Days (5 days to hatch and 28 days post-hatch)

Hardness:

126 - 136 mg/L as CaCO3)

Test temperature:

25 +/- 1 °C

pH:

8.2 - 8.4

Dissolved oxygen:

5.1 - 8.4 mg/L

Nominal and measured concentrations:

- Negative control: < LOQ
- Solvent control: < LOQ
- Nominal concentration: 20, 34, 58, 98 and 167 µg AI/L
- Measured concentration: 18, 32, 53, 86 and 170 µg AI/L

Details on test conditions:

Four replicate test chambers were maintained in each treatment and control group, with one incubation cup in each test chamber. Each incubation cup contained 20 embryos, resulting in a total of 80 embryos per treatment. At test initiation, embryos < 24 hours old were impartially distributed to incubation cups and exposed to test solution in the test chambers. After a 5-day embryo hatching period, the larvae were released into the test chambers, where exposure continued during a 28-day post-hatch juvenile growth period. Observations of the effects of test item on time to hatch, hatching success, growth and survival were used to calculate the no-observed-effect-concentration (NOEC), the lowest-observed-effect-concentration (LOEC) and the maximum acceptable toxicant concentration (MATC).

Duration:

33 d

Dose descriptor:

LOEC

Effect conc.:

32 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

18 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

33 d

Dose descriptor:

other: Maximum Acceptable Toxicant Concentration

Effect conc.:

24 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

- Hatching : Daily observations of the embryos indicated that there were no apparent differences in time to hatch between the control and treatment groups. Hatching success in the 18, 32, 53, 86 and 170 µg AI/L treatment groups was 86, 85, 90, 88 and 73 % respectively. Analysis indicated that there were no significant differences in hatching success between controls and the treatment groups.
- Mortality: Larval survival in the 18, 32, 53, 86 and 170 µg AI/L treatment groups was 84, 87, 88, 81 and 20% respectively.
- Behavioural observations: The majority of the surviving larvae in the control groups and 18, 32, 53 and 86 µg AI/L groups appeared normal during the test. There were a few sporadic observations of organisms that appeared smaller, paler or weak, were swimming erratically or lying on the bottom, or were observed with a curved spine or tail or a deformed lower jaw. There was a marked increase in observations of sublethal effects among surviving larvae in the 170 µg AI/L group, including organisms that appeared smaller, weak or lethargic, were swimming erratically, exhibiting a loss of equilibrium, or lying on the bottom, or were observed with a curved spine or a deformed lower jaw. All fish in this group exhibited signs of toxicity by Day 3 post-hatch.
- Growth: There were statistically significant differences in mean total length, wet weight and dry weight at concentrations >= 32 µg AI/L and in mean wet weight in the 18 µg AI/L treatment group.

Validity criteria fulfilled:

yes

Conclusions:

Fathead minnows (Pimephales promelas) were exposed for 33 days (5 days prior to hatch and 28 days post-hatch) to test item at mean measured concentrations of 18 to 170 μg AI/L. There were no statistically significant treatment-related effects on hatching success at any of the concentrations tested. However, there was a significant decrease in larval survival in the 170 μg AI/L treatment group, the highest concentration tested. Growth, measured in terms of total length, wet weight and dry weight, was the most sensitive biological endpoint measured in this study. Fathead minnows exposed to the test item at concentrations >= 32 μg AI/L had significantly reduced growth in comparison to the pooled controls.
Consequently, the LOEC and NOEC, based on growth, were 32 μg AI/L and 18 μg AI/L, respectively. The MATC was calculated to be 24 μg AI/L.

Executive summary:

A study was carried out according to OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test) and EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test). The study was conducted to determine the effects of the test material on the time to hatch, hatching success, survival and growth or fathead minnows, Pimephales promelas, during early life-stage development. Fathead minnow embryos were exposed to a geometric series of five test concentrations, a negative control (dilution water) and a solvent control (dimethyl formamide) under flow-through conditions. The exposure period included a 5-day embryo hatching period and a 28-day post-hatch juvenile growth period. Nominal test concentrations were 20, 34, 58, 98 and 167 µg/L. Observations of the effects of the test material were used to calculate the NOEC, LOEC and MATC. There were no statistically significant treatment-related effects on hatching success at any of the concentrations tested. However, there was a significant decrease in larval survival in the 170 µg AI/L treatment group. Growth, measured in terms of total length, wet and dry weight, was the most sensitive biological endpoint measured in this study. Fathead minnows exposed to the test material at concentrations >= 32 µg AI/L had significantly reduced growth in comparison to controls. Consequently, the LOEC and NOEC, based on growth, were 32 μg AI/L and 18 μg AI/L, respectively. The MATC was calculated to be 24 μg AI/L.

Description of key information

The key study is based on the read-across substance DDACarbonate (Wildlife International, 2004). The study was carried out according to OECD Guideline 210 and EPA OPPTS 850.1400, according to GLP. The study was conducted on fathead minnows, Pimephales promelas, during early life-stage development. Fathead minnow embryos were exposed to a geometric series of five test concentrations (nominal test concentrations of 20, 34, 58, 98 and 167 µg/L) over a 5-day embryo hatching period and a 28-day post-hatch juvenile growth period.  There were no statistically significant treatment-related effects on hatching success at any of the concentrations tested. However, there was a significant decrease in larval survival in the 170 µg AI/L treatment group. Growth was the most sensitive biological endpoint, with a significant reduction at >= 32 µg AI/L compared to controls. Consequently, the LOEC and NOEC, based on growth, were 32 μg AI/L and 18 μg AI/L, respectively. The MATC was calculated to be 24 μg AI/L.

Further supporting studies were available using the read-across substance, didecyldimethylammonium chloride (DDAC). The NOEC based on mortality, condition and growth was 0.032 mg/L (32 µg/L). The LC50 at 34 days was calculated at 0.081 mg/L (81 µg/L).

The NOEC of 0.018 mg/L based on growth is selected as the key value for the long-term fish toxicity endpoint.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.018 mg/L