Reaction product of ammonium molybdate and C12-C24-diethoxylated alkylamine (1:5-1:3)

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April to August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed according to guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection 29 June 2010

Specific details on test material used for the study:

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

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

yes

Details on preparation and application of test substrate:

Additiv 104 treatment solutions prepared in n-hexane and mixed into fine quartz sand, before allowing the solvent to evaporate in a fume hood. A ratio of 10g of sand / kg soil (dry weight equivalent) was used. Deionised water was added and mixed to the soil to achieve the required test moisture content of ca.42% of the MWHC.

Control samples were treated with the equivalent amount of deionised water and quartz sand only. Solvent control samples were treated with the equivalent amount of n-hexane and quartz sand. A bulk sample of soil was prepared for each treatment group and mixed thoroughly in a Crypto Peerless mixer to ensure even distribution of the sand and water.

Treatments were mixed in the following order: control (deionised water only), solvent control, and then the Additiv 104 treatment rate. The bulk samples of soil were then divided between the replicate test vessels so that each vessel held 500 ± 5 g dry weight of soil.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworm; E. fetida
- Source: Taken from cultures held at CEMAS.
- Age at test initiation (mean and range, SD): 6 months old (approx.). The worms were selected from an age-synchronised culture with a homogenous age structure. Individuals did not differ in age by more than 4 weeks.
- Weight at test initiation: individual wet weights of 250 - 600 mg and with visible clitellum

ACCLIMATION
- Acclimation period: 3 days in an artificial soil substrate
- Acclimation conditions (same as test or not): same as test
- Feeding: ad libitum with finely ground oatmeal (Gold Organic Oats, Mornflake Ltd., Crewe, UK) moistened with deionised water.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

56 d

Remarks:

Adult mortality and growth was assessed at Day 28 (days after treatment). Soil and cocoons were returned to the test vessels and left for a further 28 days.

Test temperature:

20+/- Celcius. Monitored throughout the study.

pH:

6.0 +/- 0.5h

Moisture:

ca. 42% of the maximum water holding capacity. Monitored by weighing a nominated replicate vessels from each treatment two or three times a week.

Details on test conditions:

With a 16 h photoperiod and an illumination of 402 - 547 Lux at the level of the test vessels.

Nominal and measured concentrations:

Nominal concentration - 1000 mg/kg dry soil weight

Reference substance (positive control):

yes

Remarks:

separate study with carbendazim

Duration:

28 d

Dose descriptor:

LD0

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: no unusual behavioural or morphology effects were observed in any treatment

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: Statistical analysis showed no statistically significant differences (p>0.05) between the Additiv 104 treatment rate of 1000 mg/kg dry soil and the solvent control treatment for juvenile production at Day 56 (Homoscedastic two-tailed t test, ToxCalc TM g

Details on results:

- Mortality at end of exposure period: None at Day 28
- Total mass of adults at beginning of test: Range of Day 0 total body weights per vessel 3.98 - 5.03 g
- Changes in body weigth of live adults (% of initial weight) at end of exposure period: Day 28: -2% to 15.9%
- No. of offspring produced: 98.1 (controls) ; 81.5 (solvent control); 74.4 (1000mg/kg soil (dw))
- No. of unhatched cocoons: 22 (controls) ; 36 (solvent control); 25 (1000mg/kg soil (dw))
- Morphological abnormalities: None
- Behavioural abnormalities: None

Reported statistics and error estimates:

Percentage mortality of the adult worms at Day 28 was determined for each of the treatments. In addition, the percentage change in mean live weight from Day 0 to Day 28 was calculated, along with the number of juveniles (and a measure of the coefficient of variation) produced by Day 56.

The mortality, live weight and juvenile production data generated were subject to statistical analysis using ToxCalc (TM)(1999). see tables in following section.

 

					Transform: Untransformed				2-Tailed
Conc-mg/Kg	Mean	N-Mean	Mean	Min	Max	CV%	N	t-Stat	Critical	MSD
D-Control	98.125	1.2040	98.125	54.000	151.000	34.082	8
S-Control	81.500	1.0000	81.500	54.000	149.000	40.446	8
1000	74.375	0.9126	74.375	46.000	102.000	29.263	8	0.510	2.145	29.953
Auxiliary Tests							Statistic		Critical		Skew	Kurt
Shapiro-Wilk's Test indicates normal distribution (p > 0.01)							0.88424		0.844		1.02626	0.9017
F-Test indicates equal variances (p = 0.30)							2.29381		8.88539
The control means are not significantly different (p = 0.33)							1.00139		2.14479
Hypothesis Test (2-tail, 0.05)							MSDu	MSDp	MSB	MSE	F-Prob	df
Homoscedastic t Test indicates no significant differences							29.9529	0.36752	203.063	780.134	0.61787	1, 14

Validity criteria fulfilled:

yes

Remarks:

Control CoV for juvenile production was greater than stated in guideline. Juvenile production in this test higher than previously seen by the test lab. Statistical analysis did not identify any outliers. This is not considered to affect validity of study.

Conclusions:

No mortality was observed in the control treatments or in the treatment rate of 1000 mg Additiv 104/kg dry soil on Day 28. As such, the EC50 for adult mortality was >1000 mg Additiv 104/kg dry soil. The NOEC for reproduction was >1000 mg Additiv 104/kg dry soil.


Statistical analysis showed there were no statistically significant differences (p>0.05) between the Additiv 104 treatment rate of 1000 mg/kg dry soil and the solvent control treatment for juvenile production at Day 56. The EC50 was determined as >1000 mg Additiv 104/kg dry soil.

Executive summary:

The chronic reproductive toxicity of Additiv 104 in earthworms has been tested according to OECD testing guideline 222. Based on a range-finding study, it was considered appropriate to carry out as a limit test at 1000 mg Additiv 104/kg dry soil.

No mortality was observed in the control treatments or in the treatment rate of 1000 mg Additiv 104/kg dry soil on Day 28. As such, the EC₅₀for adult mortality was >1000 mg Additiv 104/kg dry soil.

Statistical analysis showed there were no significant differences (p>0.05) between the Additiv 104 treatment rate of 1000 mg/kg dry soiland the solvent control treatment for adult live weight on Day 28.

Statistical analysis showed there were no statistically significant differences (p>0.05) between the Additiv 104 treatment rate of 1000 mg/kg dry soiland the solvent control treatment for juvenile production at Day 56. The EC₅₀was determined as >1000 mg Additiv 104/kg dry soil.

Description of key information

A study is available that has evaluated the effects of Additiv 104 on the survival, growth and reproduction of adult earthworms, Eisenia fetida Savigny.  The study was conducted to meet the requirements specified in OECD Guideline 222 (2004). NOEC > 1000 mg Additiv 104/kg soil (dw)

Key value for chemical safety assessment

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

1 000 mg/kg soil dw

Additional information

This study determined the effects of Additiv 104 on the survival, growth and reproduction of adult earthworms, Eisenia fetidaSavigny (Oligochaeta: Lumbricidae). The study was conducted to meet the requirements specified in OECD Guideline 222 (2004).

 

Based on the results of a range-finding test (where rates of 0.1, 1.0, 10, 100 and 1000 mg Additiv 104/kg dry soilwere evaluated) a limit test was carried out where an artificial soil substrate was treated with a single concentration of 1000 mg Additiv 104/kg dry soil. Additiv 104 treatment solutions were prepared in
n-hexane and these were thoroughly mixed into the soil substrate with a volume of deionised water to achieve a targeted final moisture content of 42% of the Maximum Water Holding Capacity (MWHC).

 

The Additiv 104 treatment was compared with a deionised water control treatment and a solvent (n-hexane ) control treatment. Eight replicate vessels were prepared for each treatment each containing 500 g (dry weight equivalent) of soil. For all treatments, the soil moisture content was maintained at approximately 42% MWHC throughout the study.

 

Ten adult E. fetida (approximately 6 months old, 250 -600 mg fresh weight and with a clitellum) were weighed and placed onto the surface of the soil substrate in each test vessel. One day after treatment (DAT), approximately 5 g of finely ground oatmeal was mixed with 10 mL of deionised water and poured onto the soil surface of each test vessel. Thereafter, food was replenished on a weekly basis during the first four weeks of the study. At 28 DAT, worms were removed and adult mortality and live weights were recorded. A final 5 g of food was incorporated into the soil substrate before being returned to the test vessels and left for a further 28 days. At Day 56, the number of juveniles hatched from the cocoons and unhatched cocoon numbers were determined.

 

The 28 DAT mortality data are summarised below. There were no statistically significant differences (p>0.05) between the Additiv 104 treatment rate of 1000 mg/kg dry soiland the solvent control treatment for live weight on Day 28. On Day 28, no unusual behavioural or morphology effects were observed in any treatment.