Quaternary ammonium compounds, C20-22-alkyltrimethyl, chlorides

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-04-04 to 2008-05-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Version / remarks:

and DIN ISO 11268-2 and BBA Guideline

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Vapour pressure: 0.0029 hPa (20 °C)
- Water solubility (under test conditions): 0.01 g/L (cmc)
- log Pow: Not determined
- pH: 5.4 (1 % a.i. in ethanol/water 1:1)
- Density: 0.84 g/cm3 (20 °C, calc.)

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): The test item was weighed out and mixed thoroughly with quartz sand. The treated quartz sand was given to the artificial soil. Demineralised water (512 mL) was added to adjust the humidity of artificial soil to a moisture of 54 % of the maximum water holding capacity. Thereafter, test medium was mixed with a long handled blender for at least three minutes and filled into test vessels.

- Controls: Artificial soil with addition of quartz sand moistened with demineralised water without test or reference item was used as control medium.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Eisenia fetida SAVIGNY 1826 (Annelida, Lumbricidae), subspecies: Eisenia fetida fetida
- Source: Own breeding (Source: Bundesanstalt für Verbraucherschutz und Landwirtschaft (BVL), Messeweg 11/12, D-38104 Braunschweig)
- Age at test initiation (mean and range, SD): earthworms (with clitellum) aged 2-12 months, not deviating by more than 1 month (4 weeks)
- Weight at test initiation (mean, range): 0.53 g, 0.3 - 0.6 g


ACCLIMATION
- Acclimation period: 2 days prior to test start to artificial soil
- Acclimation conditions (same as test or not): No food was supplied during adaptation.
- Health during acclimation (any mortality observed): healthy

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

54 d

Remarks:

Removement of adults after 28 d

Test temperature:

20 +/- 2 °C

pH:

pH-Values of the Test Media:
Application rate pH-value
[mg/kg soil DW] day 0 test end
Control 6.35 6.69
250 6.22 6.68
500 6.21 6.58
1000 6.21 6.57

Moisture:

Moisture of the Test Media:
Application rate Moisture [%] of dry weight

[mg/kg soil DW] day 0 test end
Control 40.03 45.60
250 39.90 45.71
500 42.45 44.31
1000 42.37 43.52

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Round plastic boxes with an inner diameter of 15.0 cm corresponding to a bottom surface area of 177 cm2 and a height of 14 cm; transparent and perforated lids enabled sufficient gas exchange, light input and prevented the test substrate from drying.
- Amount of soil or substrate: 600 g soil (dry weight)
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): based on OECD Guideline 207 and the EPPO Standard:
10 % peat, air dried and finely ground (2 mm);
20 % kaolin, kaolinite content > 30 %
70 % air dried quartz sand (sand with > 50 % particles size of 0.05 to 0.2 mm)
0.58 % calcium carbonate (CaCO3) to achieve a pH of 6.0 +/- 0.5
- Maximum water holding capacity (in % dry weigth): 54
- Pretreatment of soil: Two days before starting the test artificial soil was moistened to 27 % of the maximum water holding capacity. At test start appropriate volumes of demineralised water were mixed thoroughly with the soil using a kitchen aid mixer to adjust the soil to 54 % of the maximum water holding capacity. Moisture content was determined by drying a sample of the artificial soil at 105 °C and re-weighing.
- Storage (condition, duration): Room temperature, vessel covered with aluminium foil
- Stability and homogeneity of test material in the medium: see 'preparation and application'


OTHER TEST CONDITIONS
- Photoperiod: 16 h light / 8 h dark
- Light intensity: 400 - 800 lx


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Determination of lethal and sublethal effects (mortality, biomass, reproductive performance) of the test item on the earthworm species Eisenia fetida

VEHICLE CONTROL PERFORMED: no


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: Three different doses relevant to practical use were tested
- Range finding study: None

Nominal and measured concentrations:

Nominal application rates: 250 - 500 - 1000 mg/kg DW

Reference substance (positive control):

yes

Remarks:

CARBEN (Carbendazim / 502 g/L)

Duration:

54 d

Dose descriptor:

NOEC

Effect conc.:

250 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: reproduction, mortality and body weight

Remarks on result:

other: n.a.

Duration:

54 d

Dose descriptor:

LOEC

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: reproduction, mortality and body weight

Remarks on result:

other: n.a.

Details on results:

- Mortality at end of exposure period: After 28 days no significant mortality of adult earthworms was observed at all tested substance concentrationsand the control.
- Changes in body weigth of live adults (% of initial weight) at end of exposure period: Compared to the control the increase in body weight of the adult earthworms was significantly reduced at the highest test item concentration of 1000 mg/kg soil dry weight.
- No. of offspring produced: Further four weeks after removement of the adults the reproduction rate (average number of juveniles) came to 420 in the untreated control group and ranged between 211 and 391 in the test item groups. Compared to the control the reproduction was significantly reduced at the test item concentrations of 500 and 1000 mg/kg soil DW.
- No. of unhatched cocoons: Not determined
- Morphological abnormalities: After 28 days no significant pathological symptoms of adult earthworms were observed at all tested substance concentrations compared to the control.
- Behavioural abnormalities: After 28 days no significant changes in the behaviour of adult earthworms were observed at all tested substance concentrations compared to the control.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50 (reproduction) = 2.13 (1.96 - 2.30) mg a.i./kg
- Other: NOEC (reproduction, mortality, biomass) = 1 mg a.i./kg

Reported statistics and error estimates:

One Way Analysis of Variance (ANOVA, DUNNETT'S Method, Kruskal-Wallis) and a t-test was carried out to determine statistically significant differences compared to control replicates. A Normality Test and an Equal Variance Test were conducted prior to ANOVA and t-test. P-values for both Normality and Equal Variance Test are 0.05. The a-value for ANOVA test (acceptable probability of incorrectly concluding that there is a difference) is a = 0.05.

Adult mortality:

No significant mortality was observed in any of the tested doses of the test item and in the control

Behaviour and pathological symptoms:

No significant differences in pathological and behavioural symptoms between tested application rates and control were found.

Live weight:

At test start the earthworms had an individual weight of 0.30 - 0.60 g. The body weights of the earthworms in the control and in the tested doses of 250 and 500 mg/kg soil DW increased throughout the test period. In contrast, the biomass of adult earthworms at 1000 mg/kg soil DW did not increase and was significantly reduced compared to the control.

Reproduction rate:

The average number of juveniles in the untreated control group was 420 and ranged between 211 and 391 in the treated groups. Compared to the control the reproduction rate was reduced at test item concentrations of 500 and 1000 mg/kg soil DW. The coefficient of variation calculated for the reproduction of control was lower than 30 %.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, C20/22 ATQ trocken had significant effects on biomass and reproduction of adult earthworms at the test item concentrations of 500 and 1000 mg/kg soil DW.
The No Observed Effect Concentration of C20/22 ATQ trocken with respect to mortality, biomass and reproduction of Eisenia fetida fetida was observed after 8 weeks of exposure to artificial soil (containing 10 % peat) at the application rate of 250 mg/kg soil DW.

Executive summary:

Effects of C20/22 ATQ trocken (Batch no. DEG4047322) on mortality, biomass and reproduction of the earthworm species Eisenia fetida (Savigny) were determined according to the guidelines DIN ISO 11268-2 (1998), OECD 222 (2004) and BBA (1994) from April 4 to May 28, 2008 at Dr.U.Noack-Laboratorien in 31157 , Germany. The study was conducted under static conditions over 8 weeks with three different application rates of 250 – 500 - 1000 mg/kg soil dry weight (DW), which were thoroughly mixed with artificial soil containing 10 % peat. Four replicates per control and test item concentration were examined. Replicates contained 10 test organisms with an individual weight of 0.30 to 0.60 g each.

After 28 days no significant mortality and no significant pathological symptoms or changes in the behaviour of adult earthworms were observed at all tested substance concentrations compared to the control. However, compared to the control the increase in body weight of the adult earthworms was significantly reduced at the highest test item concentration of 1000 mg/kg soil dry weight. After further four weeks the reproduction rate (average number of juveniles) came to 420 in the untreated control group and ranged between 211 and 391 in the test item groups. Compared to the control the reproduction was significantly reduced at the test item concentrations of 500 and 1000 mg/kg soil DW. The validity criteria recommended by the test guidelines were fulfilled.

Table 1:     Summary of Observed Effects in the Tested Doses

Effects	C20/22 ATQ trocken
[mg test item/kg soil DW*]	250	500	1000
Mortality of adult earthworms	No	No	No
Reduction of body weight	No	No	Yes
Reduced reproduction rate (Number of juveniles)	No	Yes	Yes
Summary of all test effects	No	Yes	Yes

Table 2:     NOEC and LOEC for Reproduction, Mortality and Body Weight

C20/22 ATQ trocken
	Application rate
	[mg test item/kg soil DW*]
NOECreproduction, mortality, body weight	250
LOECreproduction, mortality, body weight	500

NOEC for C20/22 ATQ trocken with respect to mortality, body weight and reproduction of Eisenia fetida fetida, tested in artificial soil (containing 10 % peat), is 250 mg test item/kg soil DW*.

*DW: Dry Weight

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23.08.2007-06.09.2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Version / remarks:

DIN ISO 11268-1

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Vapour pressure: < 0.001 Pa (at 20°C)
- Water solubility (under test conditions): 0.01 g/L (cmc)
- Henry's law constant:
- log Pow: 3.29 (calculated based on water solubility [cmc] and n-octanol solubility)
- pKa: fully dissociated in aqueous environment
- UV absorption: none
- pH dependance on stability: none


OTHER PROPERTIES (if relevant for this endpoint)
- Fate and behaviour in the environment (where relevant to application patterns):

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Eisenia fetida fetida
- Source: own breeding (Bundesanstalt für Verbraucherschutz und Landwirtschaft (BVL), Messeweg 11/12, D-38104 Braunschweig)
- Age at test initiation (mean and range, SD): adults
- Weight at test initiation (mean and range, SD): 0.3 -0.6 g


ACCLIMATION
- Acclimation period: two days
- Acclimation conditions (same as test or not): in artificial soil
- Health during acclimation (any mortality observed):

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

14 d

Test temperature:

18 - 22 °C

pH:

5.7

Moisture:

54% of max water holding capacity

Details on test conditions:

TEST SYSTEM
- Test container (material, size): glass dishes (1.5 L)
- Amount of soil or substrate: 555g soil dry weight covered with perforated plastic film to prevent drying of test medium
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4
- No. of replicates per vehicle control: 4


SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): artificial soil according to guideline
- 10% peat (air dried and finely ground)
- 20% kaolin (kaolinite content 84 - 87%)
- 69% quartz sand (sand with >50% particles with a size of 50 - 200µm)
- 0.58% calcium carbonate

OTHER TEST CONDITIONS
- Temperature: 18 - 22°C
- Photoperiod: 16h
- Light intensity: 400 - 800lx


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- live weight of the earthworms was measured individually on day of application (day 0) and day 14.
- mortality, bahaviour and morphological changes of the earthworms were recorded 7 and 14 days after application

VEHICLE CONTROL PERFORMED: yes


TEST CONCENTRATIONS
- Spacing factor for test concentrations: limit test with 1000mg/kg dw
- Range finding study
- Test concentrations: 10 - 100 - 1000 mg/kg dw
- Results used to determine the conditions for the definitive study: 0% mortality and no reduction of live weight observed at any concentration

Nominal and measured concentrations:

limit test (nominal): 1000 mg/kg soil dry weight

Reference substance (positive control):

yes

Remarks:

2-Chloroacetamide

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality, biomass, development

Remarks on result:

other: n.a.

Details on results:

- Mortality at end of exposure period: 1 of 40
- Total mass of adults at beginning of test:
- Changes in body weigth of live adults (% of initial weight) at end of exposure period: 11% loss
- Behavioural abnormalities: no obvious pathological symptoms
- Other biological observations: none

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: (2-Chloroacetamide)
- LC50 = 53.5 mg/kg dw
- NOEC = 30.0

Table 1:           Summary of all Significant Effects

Effects	C22-trimethylammonium chloride
	1000 mg test substance/kg soil DW
Mortality [%]	0 %
Loss of body weight	20 %
Other symptoms (behaviour and pathological symptoms)	None observed
NOEC (mortality, loss of body weight, other symptoms)	> 1000

Validity criteria fulfilled:

yes

Conclusions:

No significant mortality or pathological symptoms were observed. The weight loss was < 20 % in the control group and the test item concentration 1000 mg test substance/kg soil DW during 14 days of exposure.

Executive summary:

Summary The acute effects of C22-trimethylammonium chloride (Batch: Oh 05/233) on the earthworm Eisenia fetida (Savigny 1826) were determined according to the OECD Guideline 207 and DIN ISO 11268-1 at the Dr. U.Noack-Laboratorien in 31157 Sarstedt, Germany from August 23 to September 06, 2007. The study was conducted with the nominal concentration of 1000 mg test substance/kg soil dry weight (DW) over a time of 14 days. 40 test organisms with an individual weight between 0.30 - 0.60 g, divided into four replicates, were tested per concentration and control in artificial soil as described by the guideline. No significant mortality was observed at the limit test item concentration 1000 mg test substance/kg soil DW after 14 days of exposure. No pathological symptoms or changes in behaviour of the earthworms were seen neither in the control group nor in the tested limit concentration. Mean body weight loss during the study was below 20 % in both groups and thus, biologically not significant (see Figure 1). A summary of the observations for the control and the limit test item concentration is given in Table 1. The validity criteria of the test guideline were fulfilled.

Description of key information

OECD 222

Under the conditions of this study, C20/22 ATQ trocken had significant effects on biomass and reproduction of adult earthworms at the test item concentrations of 500 and 1000 mg/kg soil DW.

The No Observed Effect Concentration of C20/22 ATQ trocken with respect to mortality, biomass and reproduction of Eisenia fetida was observed after 8 weeks of exposure to artificial soil (containing 10 % peat) at the application rate of 250 mg/kg soil DW.

 

OECD 207

No significant mortality or pathological symptoms were observed. The weight loss was < 20 % in the control group and the test item concentration 1000 mg test substance/kg soil DW during 14 days of exposure.

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for soil macroorganisms:

250 mg/kg soil dw