Quaternary ammonium compounds, (hydrogenated tallow alkyl)trimethyl, chlorides

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

From October 30, 1990 to November 20, 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.

Qualifier:

according to

Guideline:

other: U.S. EPA FIFRA 72-4 (b)

Deviations:

yes

Remarks:

but not expected to impact the results; Refer to 'Any other information on material and methods incl. tables' field for details

GLP compliance:

yes

Analytical monitoring:

yes

Vehicle:

yes

Details on test solutions:

Dilution water:
The base water used was Millipore Milli-Q™ deionized reverse osmosis treated well water.

Preparation of stock solution:
14C-test substance primary stock solutions were used to prepare test solutions for daphnid exposure. The primary stock solutions of 14C-test substance were prepared by adding measured aliquots of 14C-test substance to measured amounts of deionized water in volumetric glassware followed by shaking to ensure homogeneity. Three separate primary stock solutions were made and used during the course of the study. The 14C-test substance concentrations of the primary stock solutions were verified by liquid scintillation counting (LSC).

Test solution preparation:
Test solutions used in the study were prepared by daily adding measured aliquots of 14C-test substance primary stock solutions to dilution water in 2L volumetric flasks and bringing the volume to 2 L. Prior to adjusting the final volume to 2L, algal cells and a prepared food suspension were added to the dilution water in the 2L volumetric flask. After addition of the stocl solution and food, the test solutions were vigorously mixed to ensure homogeneity and solubilisation of the test substance.
- Test solutions were renewed daily and prepared immediately before use. At renewal, daphnids were transferred by wide-bore pipette from vessels with the 24-h test solution to the appropriate test vessels containing new test solution. In order to verify test substance concentrations in the test solutions for the deifinitive study, triplicate 1 mL aliquots were taken daily from each newly made test solutions (new solution) before transfer to the individual test vessels and from the three replicates from each test solution and control 24 h later at the time of renewal (old solutions) and counted by LSC.

Test organisms (species):

Daphnia magna

Details on test organisms:

- Daphnia magna was originally obtained from U. S. EPA’s Environmental Research Laboratory, Duluth, MN and was cultured in an environmental chamber under controlled conditions (temperature: 20±2°C; photo-period: 16-h light - 8-h dark; light intensity: 50-150 fc).
- Daphnia were cultured in 1L glass beakers (10 Daphnia per beaker) containing 800 mL of hard re-constituted laboratory water.
- Each beaker received 1.8E08 cells/L selenastrum capricornutum and 5 mL of a yeast/trout food/cerophyl suspension three times each week when the culture water was changed. Selenastrum was obtained from Battelle in-house cultures (algal slants originally obtained from the university of Texas at Austin). Purina brand trout chow, Fleischmann brand yeast and Sigma chemical company cerphyl were sources for the YTC suspension.
- Survival and reproduction of culture animals were checked each time the culture water was changed. After 28d, the adults were discarded and new cultures were started.
- 24h before the start of the test, young were removed from the beakers to ensure that only daphnids less than 24-h old would be used to start the test. the young daphnids were from 22d old cultures. There was 100% survival of cultures animals and an average of 8.6 young produced per female per reproductive day the week before the toxicity test.

Test type:

other: daily static renewal without aeration

Water media type:

freshwater

Total exposure duration:

21 d

Hardness:

160.5 mg/L as CaCO3 (124 to 228 mg/L)

Test temperature:

18.1 to 22°C

pH:

7.4 to 8.4

Dissolved oxygen:

5.6 to 9.6 mg/L (represents 64to 109% air saturation at 20°C)

Nominal and measured concentrations:

- Nominal concentrations of 0 (dilution water control), 0.5, 0.9, 1.6, 2.8 and 5 µg/L.
- Measured concentration: 0, 0.41, 0.71, 1.33, 2.31 and 4.15 µg/L

Details on test conditions:

Test system:
- Test vessel: Ten 250 mL test vessels contained 200 mL of test solution (depth of 60 mm) were used for each concentration and control.
- Number of replicates: Two for control and each test concentration.
- Number of Daphids: Seven of the ten test vessels contained an individual daphnid in each vessel while the three remaining vessels for each test concentration and control contained five daphnids each (total number of daphnids = 22 per test concentration or control).
- Aeration: No

Test conditions:
- Daphnia magna less than 24h old were exposed to mean analytical concentrations of 0, 0.41, 0.71, 1.33, 2.31 and 4.15 µg/L of the test substance in a 21d daily static-renewal test without aeration. Each concentration was run in duplicate. Test solutions were renewed daily and prepared immediately before use. Observations on daphnid behaviour, the number of live and dead (or immobilised) organisms, and the number of young produced were made at each renewal day and at test termination.
- Test temperature: 20±2°C
- Photoperiod: 16h light and 8h darkeness
- Light intensity: 103.9 fc

Observations:
- Parameters observed: The number of live and dead (or immobilized) organisms, and the number of young produced were made at each renewal day and at test termination.
- Actual measurements of the test daphnids were made in ocular units and converted to mm (1 ocular unit = 0.07 mm).
- Reproduction and growth (length) data were only collected from the seven individually exposed replicates in each exposure concentration and control. Survival data were collected for all exposure beakers.

Range-finding study:
- A 9 -d range finding experiment with Daphnia magna neonates (<24h old) were exposed to five nominal conentrations 0.1, 0.5, 1, 5 and 10 µg/L of 14C-test substance and a dilution water control.
- Two replicates each were used.
- Five neonates were placed in each test concentration and control vessel (total of 12 beakers and 60 daphnids) with a wide-bore pipette.
- Test vessels were maintained in an environmental chamber at a temperature of 20±2°C. Aeration was not used.
- For the duration of the test, observations were made on survival and reproduction of the test organisms.
- Test substance concentration were verified by using LSC.
- Based on the results from the range finding study, the definitive chronic toxicity was conducted at nominal concentrations of 0.5, 0.9, 1.6, 2.8 and 5 µg/L 14C-test substance.

Statistics:
Fisher’s Exact Test, analysis of variance, Bonferroni’s multiple comparison test

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 5 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality, reproduction and growth

Remarks on result:

other: equivalent to ≥4.15 µg a.i./L

Details on results:

Range-finding study:
- At test termination survival was ≥90% in all test concentrations and control.
- Total reproduction (live plus dead young) in the nominal 14C-test concentrations of 0 (control), 0.1, 0.5, 1, 5 and 10 µg/L was 42, 51, 58, 52, 0 and 0 young respectively.
- At test termination, a total of 2, 33 and 41 aborted eggs were found in the 1, 5, and 10 µg/L nominal test concentrations respectively.
- The overall mean of analytical values for the 0.1, 0.5, 1, 5 and 10 µg/L nominal concentrations were 0.06, 0.5, 0.96, 5.02 and 9.24 µg/L respectively.

Main study:
- The LC50 values was >4.15µg/L.
- At the end of the 21-d exposure, there was 86% survival of the test organisms in the highest measured test concentration (4.15 µg/L) and 95% or greater survival in the remaining test concentrations and in the dilution water control. Survival in all the test concentrations was not significantly different (p>0.05) from the control.
- The NOEC and LOEC for survival, therefore, were >4.15 and >4.15 µg/L of the test substance respectively.
- Reproduction of the test organisms was not significantly affected (p>0.05) in any of the test concentrations when compared to the control by ANOVA. The NOEC determined from the 21d reproductive data was ≥4.15 µg/L of the test substance.
- The growth of the test organisms at the end of the 21-d exposure period was also not significantly different (p>0.05) in any of the test concentration when compared with control using ANOVA. The NOEC and the LOEC determined from the 21d growth data was ≥4.15 and >4.15 µg/L respectively.

Reported statistics and error estimates:

Fisher’s Exact Test, analysis of variance, Bonferroni’s multiple comparison test.

Table 1. NOEC and LOEC results

Parameter		Concentration (µg/l)
Mortality	NOEC	≥4.15
	LOEC	> 4.15
Reproduction	NOEC	≥4.15
	LOEC	5.02
Growth	NOEC	≥4.15
	LOEC	> 4.15

No treatment related effects were observed in the definitive 21d study which was conducted at nominal test concentrations of 0.5, 0.9, 1.6, 2.8 and 5 µg/L. This result was surprising in light of the fact that marked effects on reproduction were observed at nominal concentrations at or above 5 µg/L in the 9 -day range-finding study.

One possible explanation for these results was that the mean analytical or actual test concentration acheived in the nominal 5 µg/L treatment group for the 9 -day range finding study was 5.02 µg/L, while the analytical test concentration for the definitive study was only 4.15 µg/L. While no effects were observed in the definitive study, the data from the range finding study clearly demonstrates effects on reproduction at test concentrations at or above 5.02 µg/L. In addition, the 48 -h LC50 in daphnids was determined at this laboratory to be 5.8 µg/L. Therefore, while an effect level was not demonstrated in the definitive study, the principle objective of the definitive study was still accomplished since the no-effect levels for the paramters of reproduction and growth were clearly defined at levels just below concentrations which have been shown to produce effects on reproduction and survival.

By supplementing the results of the 21 d definitive test with those from the 9 d range finding test, the following conclusions were drawn:

- The NOEC and LOEC for reproduction were 4.15 µg/L and 5.02 µg/L respectively.

- The MATC derived for the combined test results was 4.56 µg/L.

- The NOEC and LOEC for survival and growth are ≥4.15 and >4.15 µg/L µg/L respectively.

For further details on the results, please refer to the attachment under background material.

Validity criteria fulfilled:

not specified

Conclusions:

Based on the results of the read across study, the 21d chronic NOEC for mortality, reproduction and growth of Daphnia was considered to to be ≥ 4.15 µg a.i./L (measured)

Executive summary:

A study was conducted to determine the long-term toxicity of the read across substance, C12-16 ADBAC (98.4% radiolabeled purity) to aquatic invertebrates, according to U.S. EPA FIFRA 72-4 (b), in compliance with GLP. Based on the outcome of a 9-day range-finding study, 22 immature Daphnia magna per group were exposed to the read across substance at nominal concentrations 0.5, 0.9, 1.6, 2.8 and 5 µg/L, for 21 d under daily static-renewal conditions. The radiolabeled test concentrations were verified analytically by liquid scintillation counting. The mean measured concentrations were determined to be 0, 0.41, 0.71, 1.33, 2.31 and 4.15 µg a.i./L. Observations on daphnid behaviour, the number of immobilised organisms and the number of young produced were made at each renewal day and at test termination. No treatment related effects were observed in the definitive study at measured concentration up to 4.15 µg a.i./L. However, reproduction was clearly found to be affected at measured concentrations ≥ 5.02 µg/L in the range-finding study. The possible explanation for this result was correlated to the higher measured concentration achieved in the range-finding study (i.e., 5.02 µg/L) compared to the definitive study (i.e., 4.15 µg/L) for the nominal test concentration of 5 µg/L. While an effect level was not demonstrated in the definitive study, the principle objective of the of the definitive study was considered to be still accomplished, since the no-effect levels for the parameters of reproduction and growth were clearly defined at levels just below concentrations which showed effects in the range-finding study. Therefore, by supplementing the results of the 21-d definitive test with those from the 9-d range-finding test, the NOEC and LOEC for reproduction were considered to be at 4.15 and 5.02 µg a.i./L, respectively. In addition, based on the absence of significant effects on survival and growth parameters compared with the control group, their NOECs and LOECs were considered at ≥ 4.15 and > 4.15 µg a.i./L, respectively. The maximum acceptable toxicant concentration (MATC) derived based on these results was 4.56 µg a.i./L. Under the study conditions, the 21-d chronic NOEC for mortality, reproduction and growth of Daphnia magna was considered to be 5 µg/L (equivalent to ≥ 4.15 µg a.i./L) and the MATC was 5 µg/L (equivalent to 4.56 µg a.i./L) (McIntyre, 1992). Based on the results of the read across study, similar NOEC values can be expected for the test substance.

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

From February 03, 1995 to March 31, 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.

Qualifier:

according to

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

Cited as OECD Guideline 202, part 2 (Daphnia sp., Reproduction Test)

Deviations:

yes

Remarks:

due to analytical limitations, no concentrations tested could be analysed. Instead a solution of 1 mg/L was used under the same conditions of the test to prove stability of test concentration.

GLP compliance:

yes (incl. certificate)

Analytical monitoring:

no

Remarks:

See below section under details on analytic methods

Test organisms (species):

Daphnia magna

Details on test organisms:

Strain / Clone: Daphnia magna clone V
Source: Daphnia magna culture kept as a continuous stock at the test facility. Clone V was originally obtained from the RIZA Lelystad, NL

Breeding method: The stock was kept in 3 L beakers containing approximately 2.5 L of culture medium. The maximum number of daphnids per test
vessel was 150 for the animals being 0 to 6 d old and 80 for the animals being older than 6 d. The culture medium was batchwise refreshed at least twice a week. During culturing the animals were fed six times a week with a chlorella-suspension. The daphnid culture was kept at 19-21ºC, 8 h dark and 16 h light and light intensity of approximately 1000 lux.
Kind of food: Chlorella pyrenoidosa suspension
Feeding frequency: Six times a week
Pretreatment: No
Feeding of animals during test: Yes,
Day food (µL)
0 15
1 15
2 20
3 30
4 40
5 50
6 60
7 70
8 80
9 90
10-20 100

Test type:

semi-static

Water media type:

freshwater

Total exposure duration:

21 d

Test temperature:

Refer to the attached table under background materials

pH:

Adjusted to 7.6 by addition of aqueous NaOH-solution; Refer to the attached table under background materials

Dissolved oxygen:

Refer to the attached table under background materials

Salinity:

No information

Conductivity:

<5 µS/cm

Nominal and measured concentrations:

Nominal: 0, 0.0031, 0.0063, 0.0125, 0.025, 0.05, 0.1 or 0.2 mg/L
For stability of the nominal concentrations refer to the chemical analyses appended under 'attached background materials'

Details on test conditions:

The study was performed according to the draft OECD test guideline 202, part II: Daphnia, reproduction test, draft 6/93 with some modifications which were:
- The composition of the dilution water was according to a correction of the guideline.
- The food amount was not recalculated into TOC-content.
- The number of offspring and the number of parent animals was not counted daily, but on the Days of renewal of the test solutions only.
- The number of test vessels and the number of animals per test vessel.
- The number of offspring was tested in a two-sided Dunnett-test. 20 Daphnia magna per concentration were exposed to 0, 0.0031, 0.0063, 0.0125, 0.025, 0.05, 0.1 or 0.2 mg/L of the test substance for 22d under semi-static conditions.

The number of living offspring per parent animal per d is assessed as is the mortality of the parent animals. A NOEC and LOEC was determined for reproduction and for mortality of the parent animals.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.025 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: equivalent to 0.0125 mg a.i../L

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.05 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: equivalent to 0.025 mg a.i../L

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.05 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: equivalent to 0.025 mg a.i../L

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: equivalent to 0.05 mg a.i../L

Details on results:

NOEC mortality parent animals = 0.025 mg/L
LOEC mortality parent animals = 0.05  mg/L. 
At the next higher concentration, 0.1 mg/L all parent animals died within 48 h. Therefore there was no significant inhibition nor a stimulation effect observed at all concentrations tested.  The NOEC and LOEC for reproduction therefore amounted to respectively 0.05 mg/L and 0.1 mg/L.
Based on active substance:
NOEC mortality parent animals = 0.0125 mg a.i./L.
LOEC mortality parent animals = 0.025  mg a.i./L. 
NOEC (reproduction) = 0.025 mg a.i./L.
LOEC (reproduction) = 0.05  mg a.i./L. 

- Conclusion: 
No effects on reproduction were observed at the highest level which could be tested. The 48h-NOEC and LOEC are about a factor three higher than the 48h-NOEC and LOEC from the acute daphnia toxicity study. (NOEC 0.0125 vs 0.0048 mg a.i./L; LOEC 0.025 vs 0.0075 mg a.i./L). Although somewhat high, it is considered to be caused by normal biological variation. Besides, the NOEC and LOEC in this study are defined by a statistical significant difference of the test group to the control. Although at 0.006 mg a.i./L cumulative mortality reached 40% compared to 16% in the control, but this did not reach statistical significant level. The 48h-EC100 is in both studies the same: 0.05 mg a.i./L Irrespective of the differences in acute toxicity, this study demonstrates that reproduction capacity only appears subsequent to acute toxicity, and that the chronic NOEL therefore does not differ from the acute NOEL.

Reported statistics and error estimates:

Established on statistical evaluation of difference to controls

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of the read across study, the 21-d NOEC for survival and reproduction of Daphnia magna were 0.0125 and 0.025 mg a.i./L respectively

Executive summary:

A study was conducted to determine the long-term toxicity of the read across substance C12-16 ADBAC (50.5% active in water) to Daphnia magna according to OECD Guideline 211, in compliance with GLP. Twenty daphnids per concentration were exposed to 0, 0.0031, 0.0063, 0.0125, 0.025, 0.05, 0.1 or 0.2 mg a.i./L of read across substance for 21 days under semi-static conditions. The read across substance was suspected to adsorb to glass surfaces. In order to maintain the bioavailable concentrations in the test solutions as good as possible, the test vessels were pretreated by rinsing them with the respective test solutions. No chemical analyses of the actual concentrations of the test solutions could be performed as the test concentration were too low and no appropriate method for chemical analyses was available. However, to demonstrate the stability of the read across substance under test conditions, a stability study was performed in which a test solution of 1 mg/L was treated under test conditions for three days (the maximum period between two medium refreshments). The study showed, that the read across substance was stable under test conditions. The percentage recovery ranged between 98 and 107% through-out the 3-day test period. Therefore, all values are presented as nominal; although it cannot be excluded that the read across substance was adsorbed from the test soltions onto the walls of the test vessels, which therefore might have lowered the nominal concentrations in the test solutions. The mortality of the parent animals as compared to the control was significantly increased at 0.05 mg/L (or 0.025 mg a.i./L), which was the LOEC for mortality. Consequently, the NOEC for mortality of the parent animals amounted to 0.025 mg/L (or 0.0125 mg a.i./L). Neither significant inhibition nor significant stimulation was observed for the number of living offspring at all concentration tested up to and including 0.05 mg/L. At the next higher tested concentration of 0.1 mg/L all parent animals had died within 48 h. Therefore, the NOEC and LOEC for reproduction was established at 0.05 mg/L (i.e., 0.025 mg a.i../L) and 0.1 mg/L (i.e., 0.05 mg a.i../L) . Under the conditions of the study, the 21 -d NOEC for survival and reproduction of Daphnia magna were 0.0125 and 0.025 mg a.i./L respectively(Mark, 1995). Based on the results of the read across study, similar NOEC values can be expected for the test substance.

Reference 3

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

From 1980 to 1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.

Qualifier:

equivalent or similar to

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Principles of method if other than guideline:

Daphnia magna were exposed to six concentrations of the test substance in a 21-d static, daily renewal test. Control and isopropanol control (vehicle; IPA) groups were also evaluated. Three water types were utilised in this test: laboratory blended water (total hardness ~150 mg/L), Southwest well water (total hardness ~350 mg/L) and river water (total hardness ~300-350 mg/L). The river water, exemplifying natural surface water that received sewage effluent, was collected from the White River (Indiana) and transported for cold storage (~4°C). The test in blended water was discontinued after 14 d due to inadequate reproduction by control organisms. Mortality was monitored daily and the number of young produced in each beaker was recorded after which they were discarded. Temperature was recorded daily and pH, dissolved oxygen and hardness were determined on alternate days in control waters, both fresh and 24 h old. Daphnid 21-d length was also determined by the use of an ocular micrometer measuring from the base of the spine to the apex of the helmet. Because no statistically significant differences in daphnid length occurred as a result of exposure to increasing concentrations of the test substance, this parameter was not measured in the well water.

GLP compliance:

not specified

Analytical monitoring:

yes

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Isopropanol

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (< 24 h old)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

Water hardness value: 332 mg CaCO3/L
Upper water hardness value: 364 mg CaCO3/L

Test temperature:

Temperature value: 19.2°C
Upper temperature value: 21.2°C

pH:

pH Value: 7.5
Upper pH Value: 8.3

Dissolved oxygen:

8.8 - 9.2 mg/L

Nominal and measured concentrations:

Nominal concentrations (μg/L):
Southwest well water: 2.5, 5.0, 10.0, 20.0, 40.0 and 80.0 μg/L
River water: 74.4, 110.4, 146.4, 218.4, 290.4 and 578.4 μg/L
Measured concentrations (μg/L; values represent the geometric mean of the 0- and 24-h concentration analyses):
Southwest well water: 1.6, 3.1, 6.8, 14.6, 30.6 and 60.8 μg/L
River water: 35.7, 53.4, 68.3, 99.1, 122.3 and 309.3 μg/L

Details on test conditions:

TEST SYSTEM
- Renewal rate of test solution (frequency/flow rate): Static (daily renewal)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality was monitored daily and the number of young produced in each beaker was recorded after which they were discarded. Temperature was recorded daily and pH, dissolved oxygen and hardness were determined on alternate days in control waters, both fresh and 24 h old. Daphnid 21-day length was also determined by the use of an ocular micrometer measuring from the base of the spine to the apex of the helmet.

VEHICLE CONTROL PERFORMED: yes (isopropanol)

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

6.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Mortality and total number of young produced

Remarks on result:

other: test conducted with Southwest well water.

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

99.1 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Mortality and total number of young produced

Remarks on result:

other: test conducted with river water.

Details on results:

Please refer to the table 1 of the attached background material for detail.

Reported statistics and error estimates:

Mortality data were analysed by probit analysis to derive a 21-d NOEC value and associated 95% confidence interval. t-tests were used to analyse statistically significant differences in other tested parameters including days to first reproduction, total young production, mean brood size, and 21-d length.

Conclusions:

Based on the results of the read across study, the 21d NOEC of the test substance to Daphnia magna is considered to be 6.8 and 99.1 μg/L in Southwest well and river water, respectively.

Executive summary:

A study was conducted to determine the long-term toxicity of the read across substance, C16-18 and C18-unsatd. TMAC (48.4% active in water, radiochemical purity: 98%) to Daphnia magna under static conditions, according to OECD Guideline 211. The daphnids were exposed to six concentrations of the read across substance in a 21-day static-daily renewal test in three different water types (i.e., laboratory blended water, Southwest well water and river water). Analytical determination of the read across substance was performed. Because of the very rapid removal of the read across substance from the water column, the geometric mean of the 0, 24 and 48-hour concentrations was considered to be the overall exposure concentration in the chronic toxicity tests. The geometric mean measured concentrations were: Southwest well water: 1.6, 3.1, 6.8, 14.6, 30.6 and 60.8 μg a.i./L, river water: 35.7, 53.4, 68.3, 99.1, 122.3 and 309.3 μg a.i./L. The test in blended water was discontinued after 14 d due to inadequate reproduction by control organisms. Mortality was monitored daily and the number of young produced in each beaker was recorded. Temperature was recorded daily and pH, dissolved oxygen and hardness were determined on alternate days in control waters, both fresh and 24 h old. Daphnid 21-day length was also determined by the use of an ocular micrometer measuring from the base of the spine to the apex of the helmet.Because no statistically significant differences in Daphnid length occurred as a result of exposure to increasing concentrations of the read across substance, this parameter was not measured in the well water. The number of days until the first reproduction was similar across all groups within the Southwest well water group and river water group (i.e., 11 days and 8 - 9 days, respectively). The water chemistries remained relatively constant during the test periods. Under the conditions of the study, the 21d NOEC values were determined to be 6.8 and 99.1 μg a.i./L in Southwest well and river water, respectively. (TRS-HPV, 2001).

Description of key information

Based on the available read across studies, and in line with the C12-16 ADBAC biocides assessment report, the lowest 21d NOEC value of 4.15 µg a.i./L (initial measured) for effects on reproduction and growth of Daphnia magnahas been considered further for hazard/risk assessment.   

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:

4.15 µg/L

Additional information

Study 1:A study was conducted to determine the long-term toxicity of the read across substance, C12-16 ADBAC (98.4% radiolabeled purity) to aquatic invertebrates, according to U.S. EPA FIFRA 72-4 (b), in compliance with GLP. Based on the outcome of a 9-day range-finding study, 22 immature Daphnia magna per group were exposed to the read across substance at nominal concentrations 0.5, 0.9, 1.6, 2.8 and 5 µg/L, for 21 d under daily static-renewal conditions. The radiolabeled test concentrations were verified analytically by liquid scintillation counting. The mean measured concentrations were determined to be 0, 0.41, 0.71, 1.33, 2.31 and 4.15 µg a.i./L. Observations on daphnid behaviour, the number of immobilised organisms and the number of young produced were made at each renewal day and at test termination. No treatment related effects were observed in the definitive study at measured concentration up to 4.15 µg a.i./L. However, reproduction was clearly found to be affected at measured concentrations ≥ 5.02 µg/L in the range-finding study. The possible explanation for this result was correlated to the higher measured concentration achieved in the range-finding study (i.e., 5.02 µg/L) compared to the definitive study (i.e., 4.15 µg/L) for the nominal test concentration of 5 µg/L. While an effect level was not demonstrated in the definitive study, the principle objective of the of the definitive study was considered to be still accomplished, since the no-effect levels for the parameters of reproduction and growth were clearly defined at levels just below concentrations which showed effects in the range-finding study. Therefore, by supplementing the results of the 21-d definitive test with those from the 9-d range-finding test, the NOEC and LOEC for reproduction were considered to be at 4.15 and 5.02 µg a.i./L, respectively. In addition, based on the absence of significant effects on survival and growth parameters compared with the control group, their NOECs and LOECs were considered at ≥ 4.15 and > 4.15 µg a.i./L, respectively. The maximum acceptable toxicant concentration (MATC) derived based on these results was 4.56 µg a.i./L.Under the study conditions, the 21-d chronic NOEC for mortality, reproduction and growth of Daphnia magna was considered to be 5 µg/L (equivalent to ≥ 4.15 µg a.i./L) and the MATC was 5 µg/L (equivalent to 4.56 µg a.i./L) (Mclntyre, 1992).

Study 2:A study was conducted to determine the long-term toxicity of the read across substance C12-16 ADBAC (50.5% active in water) to Daphnia magna according to OECD Guideline 211, in compliance with GLP. Twenty daphnids per concentration were exposed to 0, 0.0031, 0.0063, 0.0125, 0.025, 0.05, 0.1 or 0.2 mg a.i./L of read across substance for 21 days under semi-static conditions. The read across substance was suspected to adsorb to glass surfaces. In order to maintain the bioavailable concentrations in the test solutions as good as possible, the test vessels were pretreated by rinsing them with the respective test solutions. No chemical analyses of the actual concentrations of the test solutions could be performed as the test concentration were too low and no appropriate method for chemical analyses was available. However, to demonstrate the stability of the read across substance under test conditions, a stability study was performed in which a test solution of 1 mg/L was treated under test conditions for three days (the maximum period between two medium refreshments). The study showed, that the read across substance was stable under test conditions. The percentage recovery ranged between 98 and 107% through-out the 3-day test period. Therefore, all values are presented as nominal; although it cannot be excluded that the read across substance was adsorbed from the test soltions onto the walls of the test vessels, which therefore might have lowered the nominal concentrations in the test solutions. The mortality of the parent animals as compared to the control was significantly increased at 0.05 mg/L (or 0.025 mg a.i./L), which was the LOEC for mortality. Consequently, the NOEC for mortality of the parent animals amounted to 0.025 mg/L (or 0.0125 mg a.i./L). Neither significant inhibition nor significant stimulation was observed for the number of living offspring at all concentration tested up to and including 0.05 mg/L. At the next higher tested concentration of 0.1 mg/L all parent animals had died within 48 h. Therefore, the NOEC and LOEC for reproduction was established at 0.05 mg/L (i.e., 0.025 mg a.i../L) and 0.1 mg/L (i.e., 0.05 mg a.i../L). Under the conditions of the study, the 21-d chronic NOEC for survival and reproduction of Daphnia magna were 0.0125 and 0.025 mg a.i./L (or 12.5 and 25 µg a.i./L) respectively (Mark, 1995).

Based on the above studies, same effect levels were concluded in the biocide assessment report on C12-16 ADBAC by RMS Italy. They further discussed that due to the differences in the study methodologies and measurements, a geometric mean of the two chronic values was not calculated. Instead the lowest value 21d NOEC ≥ 0.00415 mg a.i./L was selected, which was based on mean measured concentration and was more protective, especially considering the acute toxicity data (ECHA biocides assessment report, 2015).

Study 3: A study was conducted to determine the long-term toxicity of the read across substance, C16-18 and C18-unsatd. TMAC (48.4% active in water, radiochemical purity: 98%) to Daphnia magna under static conditions, according to OECD Guideline 211. The daphnids were exposed to six concentrations of the read across substance in a 21-day static-daily renewal test in three different water types (i.e., laboratory blended water, Southwest well water and river water). Analytical determination of the read across substance was performed. Because of the very rapid removal of the read across substance from the water column, the geometric mean of the 0, 24 and 48-hour concentrations was considered to be the overall exposure concentration in the chronic toxicity tests. The geometric mean measured concentrations were: Southwest well water: 1.6, 3.1, 6.8, 14.6, 30.6 and 60.8 μg a.i./L, river water: 35.7, 53.4, 68.3, 99.1, 122.3 and 309.3 μg a.i./L. The test in blended water was discontinued after 14 d due to inadequate reproduction by control organisms. Mortality was monitored daily and the number of young produced in each beaker was recorded. Temperature was recorded daily and pH, dissolved oxygen and hardness were determined on alternate days in control waters, both fresh and 24 h old. Daphnid 21-day length was also determined by the use of an ocular micrometer measuring from the base of the spine to the apex of the helmet.Because no statistically significant differences in Daphnid length occurred as a result of exposure to increasing concentrations of the read across substance, this parameter was not measured in the well water. The number of days until the first reproduction was similar across all groups within the Southwest well water group and river water group (i.e., 11 days and 8 - 9 days, respectively). The water chemistries remained relatively constant during the test periods. Under the conditions of the study, the 21d NOEC values were determined to be 6.8 and 99.1 μg a.i./L in Southwest well and river water, respectively. (TRS-HPV, 2001).

Overall, based on the available read across studies, and in line with the C12 -16 ADBAC biocides assessment report, the lowest 21d NOEC value of 4.15 µg a.i./L (initial measured)for effects on reproduction and growth of Daphnia magna has been considered further for hazard/risk assessment. ​