6,6'-di-tert-butyl-4,4'-butylidenedi-m-cresol

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:

16 February 2017 to 13 April 2017

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:

OECD Guideline for the Testing of Chemicals, “Earthworm Reproduction Test (Eisenia fetida / Eisenia andrei)”, No.: 222, adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Analytical monitoring:

no

Vehicle:

yes

Remarks:

fine quartz sand

Details on preparation and application of test substrate:

FORMULATION
Immediately before commencement of the test, the test item was thoroughly mixed with ~10 g fine quartz sand, applied with this mixture to artificial soil then moistened using a laboratory mixer. The control was similarly prepared using sand.
In order to obtain a better distribution of the test item fine quartz sand mixtures in the soil the test substrate was prepared by mixing the amount of soil needed for two replicates (1288.7 g dry weight) in one step.
The test item concentrations were processed shortly before treatment as follows (reported in table form – see “Any other information” for details)/

TEST SUBSTRATE
The availability of lipophilic test item (ingredients) in soil and therefore their toxicity to earthworms depends on the organic matter content of the soil. For chemicals with log Pow > 2, sorption is expected to be linearly, related to soil organic matter content. With respect to the properties of the test item (log Pow >2) 5 % instead of 10 % peat was used, considering the influence on bioavailability (OECD 222).
The partition coefficient (log Pow) of Active ingredient (6,6'-di-tert-butyl-4,4'-butylidenedim- cresol): 6.4 (at 20ºC, based on information provided by the Sponsor).
Artificial soil was prepared, as follows:
5 % sphagnum peat (as close to pH 5.5-6.0 as possible, no visible plant remains, finely ground, dried to measured moisture content)
20 % kaolin clay (kaolinite content above 30 %)
75 % industrial sand (fine sand is dominant with more than 50 % of the particles between 50 and 200 μm)
The air-dry constituents were blended in the correct proportions and mixed thoroughly with deionised water. The final water content was equivalent to about 60 % of the maximum WHC. The determination of pH value was done according to ISO 10390.
The pH values of the treated artificial soil were determined at the beginning, and at the end of the test.
The maximal water holding capacity (WHC) of the artificial soil was determined according to OECD 222 ANNEX 2.
The moistened soil was divided in test containers corresponding with the number of test concentrations and controls.
Soil moisture content was determined at the beginning and at the end of the test by SARTORIUS Moisture Analyzer.
The water content of the artificial soil before moisturing was 1.62 g / 100 g dry soil. The maximal water holding capacity of the artificial soil was 45.50 g water / 100 g soil dry weight.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

Taxonomic Group: Earthworms (Annelida: Oligochaeta)
Species: Eisenia fetida (Savigny 1826)
Life Stage: adults, adequately fed, healthy, disease-free colonies
Age: about 10 months (not differing in age by more than 4 weeks) with clitellum
Body Weight: 302 - 599 mg, including gut contents
Origin: BASF SE Crop Protection Division, Limburgerhof
Strain code: VT BASF 05 09 165 – 166 - 167 (in CiToxLAB Hungary Ltd.)
Bred under standardised conditions (see OECD 207) in the Ecotoxicological Laboratory of CiToxLAB Hungary Ltd., Veszprém-Szabadságpuszta
Acclimatisation: 1 day, in artificial soil, under test environmental conditions (day–1 – day 0)
Number of animals: There were 40 earthworms at each concentration and 40 in the untreated control; each were divided into 4 replicates (10 worms/replicate)

Housing and Environmental Conditions
The earthworms were conditioned for about 24 hours in the test substrate. Then they were housed in appropriate plastic containers (with ~1L volume and ~200 cm2 area), filled with the moist artificial soil (the amount of the soil: 763.8 g/test container corresponded to 600 g soil dry weight). These plastic containers were covered with transparent and punctured lid to allow gas exchange.
The test containers were identified with numbers (study code, test groups, replicates). The test containers were held in a climate chamber under continuous lighting with at a light intensity of about 482 lux. The temperature ranged from 19.0 °C – 21.0 °C. In each container there were 10 earthworms.
At the start of the test the moisture content of the test medium was adjusted to about 60 % of the maximum WHC which corresponded about 21.4 % of the soil dry weight. The moisture content was checked in two replicates of all concentration levels. The average water content was 25.93 % of soil dry weight at the beginning of the study.
At the end of the test it was determined in the same replicates and found to be 25.76 % of soil dry weight. The water content and the pH values of the samples measured on day 0 and day 56 of the experiment, it was 6.39 – 6.59 at test initiation and 6.24 - 6.64 at the end of the test (and the maximal water holding capacity measured on day -1).

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

56 d

Post exposure observation period:

28d incubation period

Test temperature:

19.0°C – 21.0 °C

pH:

6.24 - 6.64

Moisture:

25.76 %

Details on test conditions:

Experimental Design
The following concentrations of LOWINOX® 44B25 were used in the study: 62.5; 125; 250; 500 and 1000 mg Test Item/kg soil dry weight.
Four replicates, each containing 10 worms were included for each exposure concentration and control.

Duration of the Test
1. Pre-exposure period: 1 day
2. Treatment
-Exposure period: 28 days
-Incubation period: 28 days

Treatment
The earthworms were conditioned for about 24 hours in the artificial soil (day -1 – day 0).
Immediately before the start of the test, the appropriate amounts of the test item were mixed thoroughly with the artificial soil so that the moisture (water content) of the soil was approximately 60 % of the maximum WHC, which corresponded about 21.4 % of the soil dry weight.
Care was taken to avoid any contamination of the control. The test item concentrations were applied in increasing order, thus minimizing the risk of contamination.
On day 0, after washing and drying gently, the individual weight of each worm was determined, and then worms were exposed to the treated artificial soil.
For each concentration and the controls, four replicate test containers were used each containing 600 g dry weight of artificial soil.

Feeding
One day after application of the test item to the soil the earthworms were fed with approximately 5 g air-dried finely ground cow manure. It was spread on the soil surface of the containers and moistened with the amount of missing water. Food was provided once a week during the exposition period, on days: 1, 7, 14, 21 and 28. The amounts of cow manure added depended on the amounts of food consumed by the worms. The water content of the test substrate was controlled weekly during the test by weighing the containers and replenishing the missing water.

OBSERVATIONS
The following observations were done:
Mortality:
During the exposure period (days 0-28) without disturbing the soil only one dead worm was observed in the control and in the lowest test item treated group of 62.5 mg/kg soil dry weight which was considered to be due to natural causes rather than a toxic effect and this mortality was only 2.5 % in this test group.
On day 28 of the experiment the adult worms were removed from the soil and the number of living adult worms were recorded. Mortality was assessed by emptying the test medium from each replicate onto a suitable tray and sorting worms from the medium. Earthworms were considered dead when they failed to respond to gentle stimulation at the front end. Due to rapid decomposition under test conditions, missing earthworms were considered dead.
Adult worms were humanely euthanized, by rapid freezing at -80°C.

Clinical Symptoms:
During the exposure period (days 0-28) no symptoms of earthworms were observed without disturbing the soil.
On day 28 of the experiment the surviving adult worms in each test container were removed from the soil, and no unusual symptoms were observed.
Biomass:
The live weight of the adult worms in every test container was determined on days 0 and 28 of the experiment after they had been removed from the substrate and been gently washed and dried.
Food consumption:
The food consumed was estimated with visual checking. When food consumption was low, feeding was reduced. The quantity of food applied was recorded for each container on days: 1, 7, 14, 21 and 28.
Number of juvenile worms:
On day 56 of the experiment the juvenile worms hatched from cocoons were removed from the soils using water bath at a temperature 50-60 °C and counted, then humanely euthanized, by rapid freezing at -80°C.
Observation of environmental parameters:
At the start of the test, the moisture content of the test medium was adjusted to approximately 60 % of the maximum WHC. At the start and at the end of the test it was determined.
The pH of the control and treated soil samples was measured on day 0 and 56 of the study.
The temperature was measured continuously (using a min/max thermometer) and recorded within the climate chamber on weekdays during the test.
The light intensity in the climate chamber was checked at the start of the test and at each moving of the test units to another climate chamber.

Nominal and measured concentrations:

Nominal concentrations: Control, 62.5, 125; 250; 500 and 1000 mg Test Item/kg soil dry weight.

Reference substance (positive control):

yes

Remarks:

Carbendazim

Key result

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

Key result

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

56 d

Dose descriptor:

other: NOAEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

MORTALITY
Mortality of the earthworms was assessed between days 1 and 28 of the experiment without disturbing the soil and after 28 days when the parent worms were removed from the soil.
During the exposure period (days 0-28) without disturbing the soil only one dead worm was observed in the lowest test item treated group of 62.5 mg/kg soil dry weight, which was considered to be due to natural causes rather than a toxic effect and this mortality was only 2.5 % in this test group.
No mortality of adult (parent) worms was observed in any other treated variants.
The LC50 of LOWINOX® 44B25 could not be calculated due the lack of significant effects.

CHANGES IN BIOMASS
The biomass of the untreated control group increased for 3.49% and increase could be observed in the whole tested concentration range as well. The biomass development was not statistically significantly inhibited at the whole tested concentration range of 62.5 - 1000 mg/kg soil dry weight (Dunnett’s Test, α=0.05).

FOOD CONSUMPTION
Statistical analysis of feeding activity of the earthworms was not performed as food consumption was the same at the all of the tested concentration levels as in the control.

NUMBER OF JUVENILES
In the control the mean number of offspring was 184.75 juveniles per replicate.
The coefficient of variance of reproduction in control was: 13.93 %.
The number of juveniles and the percentage mean of juveniles were not statistically significant compared to the control group at the whole test item concentration range of 62.5 - 1000 mg/kg soil dry weight.
The EC50, based on the reproduction rate (juvenile numbers) of Eisenia fetida was determined to be higher than 1000 mg/kg soil dry weight, the highest tested concentration rate in the study.

Results with reference substance (positive control):

The test item Carbendazim (Active ingredient: 99.3 %) has adverse effect on the reproduction of earthworms (Eisenia fetida).
EC50 (56 day): 1.75 mg Carbendazim/kg soil dry weight (95 % confidence limits: 1.62 - 1.89 mg a.i./kg soil dry weight)
LC50 (28 day): > 6.0 mg Carbendazim/kg soil dry weight
NOEC (28 day): 6.0 mg Carbendazim/kg soil dry weight (based on the adult mortality)
NOEC (28 day): 3.0 mg Carbendazim/kg soil dry weight (based on the biomass development)
NOAEC (56 day): 0.75 mg Carbendazim/kg soil dry weight (based on reproduction)
LOAEC (56 day): 1.5 mg Carbendazim/kg soil dry weight

Reported statistics and error estimates:

Mortality was 2.5% in the control and the mean reproduction in control was 184.75 juveniles per container. The coefficient of variance of reproduction in control was: 13.93 %. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Earthworm Mortality after 28 Days at Different Concentrations of LOWINOX® 44B25

Concentration of Test Item [mg/kg soil dry weight]	Number of dead worms / replicates				Mean [%]	SD
	1	2	3	4
Control	0	0	0	1	2.5	5.0
62.5	0	0	0	1	2.5^n.s.	5.0
125	0	0	0	0	0.0^n.s.	0.0
250	0	0	0	0	0.0^n.s.	0.0
500	0	0	0	0	0.0^n.s.	0.0
1000	0	0	0	0	0.0^n.s.	0.0

SD: Standard deviation

n.s.: statistically not significant compared to the control (Dunnett’s Test α=0.05).

 

Weight Changes [%] of the Parent Earthworms at Different Concentrations of LOWINOX® 44B25

Concentration of Test Item [mg/kg soil dry weight]	Weight change [%]				Mean [%]	SD
	1	2	3	4
Control	5.88	2.40	3.77	1.91	3.49	1.78
62.5	5.57	1.72	0.93	8.20	4.10^n.s.	3.40
125	-3.91	13.86	10.05	13.33	8.33^n.s.	2.07
250	-1.21	6.36	7.04	11.48	5.91^n.s.	5.26
500	11.67	5.96	12.51	17.35	11.87^n.s.	4.67
1000	10.65	4.33	8.37	15.72	9.77^n.s.	4.75

SD: Standard deviation

n.s.: statistically not significant compared to the control (Dunnett’s Test, α=0.05)

 

Total Amount of Food (g Dry Cow Manure) Added per Variant (Average of Four Replicates) during the Course of the Test

Concentration of Test Item [mg/kg soil dry weight]	Total amount of dry cow manure added per variant (average of four replicates) [g]
Control	25.00
62.5	25.00
125	25.00
250	25.00
500	25.00
1000	25.00

 

The Number of Juveniles at Different Concentrations of LOWINOX® 44B25

Concentration of Test Item [mg/kg soil dry weight]	Number of juveniles / Replicates				Mean	SD
	1	2	3	4
Control	193	216	156	174	184.75	25.73
62.5	189	214	152	172	181.75^n.s.	26.29
125	168	163	203	160	173.50^n.s.	19.94
250	151	178	171	180	170.00^n.s.	13.24
500	178	181	168	144	167.75^n.s.	16.78
1000	144	194	165	164	166.75^n.s.	20.58

SD: Standard deviation

n.s.: statistically not significant compared to the control (Dunnett’s Test, α=0.05)

 

TEST CONDITIONS

 

Water content, Water Holding Capacity and pH of the Artificial Soil Samples

Concentration [mg/kg]	Water content [g/100 g soil dry weight]				Water content [g/100 g] of WHC^1)		pH (mean)
	Day 0	Mean [day 0]	Day 56	Mean [day 56]	Day 0	Day 56	Day 0	Day 56
Control	26.12	25.93	26.20	25.76	56.99	56.61	6.48	6.32
62.5	26.03		26.84				6.44	6.48
125	26.11		26.31				6.46	6.49
250	25.65		24.24				6.40	6.54
500	25.87		26.72				6.54	6.56
1000	25.81		24.23				6.55	6.34

WHC: Water holding capacity

1) max. WHC of the test substrate = 45.50 g / 100 g soil dry weight

 

Temperature

Day	Temperature [°C]		Day	Temperature [°C]		Day	Temperature [°C]
	Min.	Max.		Min.	Max.		Min.	Max.
-1	20.2	20.9	19	19.3	20.4	40	19.2	20.5
0	19.9	20.6	20	19.3	20.3	41	19.1	20.5
1	19.7	20.2	21	19.2	20.2	42	19.1	20.6
4	19.8	20.7	22	19.1	20.2	43	19.2	20.7
5	19.5	20.2	25	19.1	20.2	46	19.0	21.0
6	19.7	20.2	26	19.3	20.2	47	19.2	20.8
7	19.5	20.2	28	19.7	20.6	48	19.2	20.7
8	19.7	20.5	29	19.2	20.5	49	19.3	20.9
11	19.1	20.1	32	19.1	20.4	50	19.0	20.6
12	19.3	20.2	33	19.1	20.4	53	19.4	20.5
13	19.3	20.2	34	19.1	20.6	54	19.4	20.6
14	19.3	20.3	35	19.0	20.3	55	19.5	20.5
15	19.3	20.4	36	19.0	20.4	56	19.3	20.6
18	19.1	20.2	39	19.1	20.5

 

BIOMASS DEVELOPMENT, FEEDING, JUVENILES

 

Individual Worm Weights

Concentration [mg/kg]	Replicate	Day	Individual worm weights [mg]										Mean	SD
Control	(1)	0 28	479 460	500 654	405 587	378 347	465 447	432 333	396 362	374 412	331 444	374 331	413.4 437.7	54.09 108.86
	(2)	0 28	547 334	412 569	507 508	337 311	444 443	354 499	359 487	467 403	476 401	386 437	428.9 439.2	70.72 80.09
	(3)	0 28	333 450	457 619	474 456	457 425	501 374	530 543	356 356	379 406	359 536	509 354	435.5 451.9	72.19 88.82
	(4)	0 28	432 611	338 483	379 341	532 355	536 621	409 403	397 450	359 280	523 552	561 -	446.6 455.1	83.22 121.68
62.5	(1)	0 28	566 427	454 466	360 596	412 452	463 515	391 531	424 484	561 415	351 376	418 383	440.0 464.5	74.15 69.23
	(2)	0 28	355 411	567 497	556 427	402 359	454 399	454 502	347 398	429 364	547 525	317 522	442.8 450.4	90.62 70.04
	(3)	0 28	386 378	350 503	494 556	536 403	390 565	444 393	442 400	444 333	526 327	309 503	432.1 436.1	74.25 88.26
	(4)	0 28	386 429	369 548	481 497	406 515	473 298	533 497	530 305	318 628	312 519	542 -	435.0 470.7	88.23 109.21
125	(1)	0 28	562 412	450 385	487 556	453 531	504 392	410 382	352 295	513 469	303 253	312 501	434.6 417.6	88.38 98.18
	(2)	0 28	331 785	407 520	469 489	342 349	441 469	505 302	403 586	475 527	319 392	484 336	417.6 475.5	68.15 143.52
	(3)	0 28	330 428	428 436	349 493	403 488	390 423	394 478	476 415	378 467	526 382	357 427	403.1 443.6	59.92 35.99
	(4)	0 28	327 457	449 628	475 660	444 468	599 374	441 335	559 539	346 568	380 390	452 649	447.2 506.8	85.37 119.23
250	(1)	0 28	374 419	344 444	374 324	491 332	431 478	486 463	503 512	428 453	457 449	323 386	421.1 416.0	54.66 53.48
	(2)	0 28	480 580	372 450	569 422	541 391	391 514	455 631	338 419	398 429	321 360	553 503	441.8 469.9	91.14 85.88
	(3)	0 28	477 232	460 470	389 337	302 422	364 585	437 511	552 399	468 580	359 502	513 587	432.1 462.5	77.31 116.83
	(4)	0 28	478 565	394 499	468 530	466 433	375 472	425 508	356 307	455 386	363 520	403 443	418.3 466.3	46.36 76.74
500	(1)	0 28	379 508	466 427	436 568	352 520	528 477	358 601	563 490	543 516	383 334	364 441	437.2 488.2	82.54 75.39
	(2)	0 28	518 433	371 530	375 449	329 488	344 440	385 407	312 476	558 461	561 383	389 322	414.2 438.9	94.59 58.23
	(3)	0 28	421 436	481 480	437 500	360 501	390 373	443 431	455 523	423 171	333 658	326 505	406.9 457.8	52.67 125.32
	(4)	0 28	530 458	428 421	374 475	379 713	396 503	518 587	350 437	440 479	332 619	559 361	430.6 505.3	79.85 104.98
1000	(1)	0 28	436 542	409 561	452 390	391 418	378 471	507 211	420 503	351 480	390 607	350 336	408.4 451.9	48.06 117.52
	(2)	0 28	426 337	388 383	393 442	460 389	390 418	308 465	385 452	548 529	390 404	335 378	402.3 419.7	66.19 54.42
	(3)	0 28	414 461	437 376	488 785	395 320	359 360	409 368	446 444	466 577	375 526	582 520	437.1 473.7	64.60 137.75
	(4)	0 28	364 345	422 448	368 631	361 383	395 401	338 426	407 423	410 494	354 502	480 459	389.9 451.2	41.95 79.37

 

 

Amount of Food Added (g Cow Manure Dry Weight per Replicate) to the Different Treatment Groups during the Test

Concentration [mg/kg]	Replicate	Day						Total	Mean
		1	7	14	21	28	35-49
Control	(1)	5	5	5	5	5	0	25	25.0
	(2)	5	5	5	5	5	0	25
	(3)	5	5	5	5	5	0	25
	(4)	5	5	5	5	5	0	25
62.5	(1)	5	5	5	5	5	0	25	25.0
	(2)	5	5	5	5	5	0	25
	(3)	5	5	5	5	5	0	25
	(4)	5	5	5	5	5	0	25
125	(1)	5	5	5	5	5	0	25	25.0
	(2)	5	5	5	5	5	0	25
	(3)	5	5	5	5	5	0	25
	(4)	5	5	5	5	5	0	25
250	(1)	5	5	5	5	5	0	25	25.0
	(2)	5	5	5	5	5	0	25
	(3)	5	5	5	5	5	0	25
	(4)	5	5	5	5	5	0	25
500	(1)	5	5	5	5	5	0	25	25.0
	(2)	5	5	5	5	5	0	25
	(3)	5	5	5	5	5	0	25
	(4)	5	5	5	5	5	0	25
1000	(1)	5	5	5	5	5	0	25	25.0
	(2)	5	5	5	5	5	0	25
	(3)	5	5	5	5	5	0	25
	(4)	5	5	5	5	5	0	25

 

Validity criteria fulfilled:

yes

Conclusions:

The validity criteria of the study are met.
The test item LOWINOX® 44B25 has no adverse effect on the reproduction of earthworms (Eisenia fetida).
EC50 (56 day): > 1000 mg Test Item/kg soil dry weight
LC50 (28 day): > 1000 mg Test Item/kg soil dry weight
NOEC (28 day): 1000 mg Test Item/kg soil dry weight (based on the adult mortality and biomass development)
NOAEC (56 day): 1000 mg Test Item/kg soil dry weight (based on reproduction)

Executive summary:

Test item:

LOWINOX® 44B25; [Batch No.: W4B5A0009]

 

Test species:

Earthworm (Eisenia fetida), from in-house culture, adult worms (with clitellum and weight 302 - 599 mg), less than one year old (~ 10 month).

 

Test design:

Different concentrations of the test item were mixed homogeneously into the soil which was filled in plastic containers before the earthworms were introduced on top of the soil; 5 concentrations and an untreated control; 4 replicates/concentration (4 replicates/untreated control group) with 10 worms each. After 28 day: assessment of worm mortality, behavioural effects and measurement of weight change as sub lethal parameter. After 56 days: assessment of the number of juveniles. The test was performed according to guideline OECD 222 (2016) and ISO 11268, part 2 (1998).

 

Endpoints:

Assessment of mortality, behavioural effects, weight change after 28 days and the number of juveniles (reproduction) after 56 days.

 

Reference item:

Carbendazim (active ingredient content: 99.3 %); Effects have been evaluated in a separate study. Study Code: 16/280-211G. Test facility as in this study.

 

Test concentrations:

Control, 62.5, 125; 250; 500 and 1000 mg Test Item/kg soil dry weight.

 

Test conditions:

Artificial soil according to OECD 222; pH 6.39 – 6.59 at test initiation, at the end of the test pH 6.24 - 6.64; average water content at test initiation 25.93 % (of soil dry weight; 56.99 % of the max. water holding capacity), 25.76 % at test termination (of soil dry weight; 56.61 % of the max. water holding capacity); temperature: 19.0°C – 21.0 °C; continuous lighting at a light intensity of about 482 lux.

 

Statistics:

Calculation of treatment means and standard deviations, analysis of variance (ANOVA) followed by Dunnett’s Test. The EC50 value (the test item concentration which causes 50 % reduction in the reproductive output) of the test item could not be calculated (due to the slight effects observed) and therefore determined directly from the raw data. The LC50 value of the test item could not be calculated due to the slight effects observed.

 

Findings:

One single dead earthworm was noted in the control and in the lowest test item treated group of 62.5 mg/kg soil dry weight during the experiment which was considered to be due to natural causes rather than a toxic effect and this mortality was only 2.5 % in these test groups.

Mortality of adult (parent) worms was not observed in any other concentration level after 28 days of exposure.

The food consumption was the same at all of the treatment levels as in the control group during the experiment.

The mean number of juveniles in the control group was 184.75 per replicate. The number of juveniles and as well as the percentage mean of juveniles was not statistically significant compared to the control group at the whole tested concentration range of 62.5 - 1000 mg/kg soil dry weight.

The EC50, based on the reproduction rate (juvenile numbers) of Eisenia fetida was determined to be higher than 1000 mg/kg soil dry weight, the highest tested concentration rate in the study.

There was an increase in earthworm biomass in the untreated control (3.49 %) and in all test item treated group. The biomass development was not statistically significantly inhibited at the whole tested concentration range of 62.5 - 1000 mg/kg soil dry weight (Dunnett’s Test, α=0.05).

The biological results of the test are summarised in the table below:

 

Summary Effect of LOWINOX® 44B25 on Earthworm (Eisenia fetida)

Mortality, Biomass Development and Reproduction in a Reproduction Study

Concentration of Test Item [mg/kg soil dry weight]	Control	32.5	125	250	500	1000
Mortality [%]-28 days	2.5 -	2.5 n.s.	0.0 n.s.	0.0 n.s.	0.0 n.s.	0.0 n.s.
Weight change [%]-28 days	3.49 -	4.10 n.s.	8.33 n.s.	5.91 n.s.	11.87 n.s.	9.77 n.s.
Number of juveniles-56 days	184.75 -	181.75 n.a.	173.50 n.s.	170.00 n.s.	167.75 n.s.	166.75 n.s.
Reproduction in % of control-56 days	100.00 -	98.38 n.s.	93.91 n.s.	92.02 n.s.	90.80 n.s.	90.26 n.s.

n.s. : statistically not significant compared to the control (Dunnett’s Test, α=0.05).

 

CONCLUSION

The validity criteria of the study are met.

The test item LOWINOX® 44B25 has no adverse effect on the reproduction of earthworms (Eisenia fetida).

EC50 (56 day): > 1000 mg Test Item/kg soil dry weight

LC50 (28 day): > 1000 mg Test Item/kg soil dry weight

NOEC (28 day): 1000 mg Test Item/kg soil dry weight

(based on the adult mortality and biomass development)

NOAEC (56 day): 1000 mg Test Item/kg soil dry weight

(based on reproduction)

Description of key information

The test item LOWINOX® 44B25 has no adverse effect on the reproduction of earthworms (Eisenia fetida).

EC50 (56 day): > 1000 mg Test Item/kg soil dry weight

LC50 (28 day): > 1000 mg Test Item/kg soil dry weight

NOEC (28 day): 1000 mg Test Item/kg soil dry weight

(based on the adult mortality and biomass development)

NOAEC (56 day): 1000 mg Test Item/kg soil dry weight

(based on reproduction)

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

1 000 mg/kg soil dw

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

1 000 mg/kg soil dw

Additional information

Different concentrations of the test item were mixed homogeneously into the soil which was filled in plastic containers before the earthworms were introduced on top of the soil; 5 concentrations and an untreated control; 4 replicates/ concentration (4 replicates/untreated control group) with 10 worms each. After 28 day: assessment of worm mortality, behavioural effects and measurement of weight change as sub lethal parameter. After 56 days: assessment of the number of juveniles. The test was performed according to guideline OECD 222 (2016) and ISO 11268, part 2 (1998).

 

Findings:

One single dead earthworm was noted in the control and in the lowest test item treated group of 62.5 mg/kg soil dry weight during the experiment which was considered to be due to natural causes rather than a toxic effect and this mortality was only 2.5 % in these test groups.

Mortality of adult (parent) worms was not observed in any other concentration level after 28 days of exposure.

The food consumption was the same at all of the treatment levels as in the control group during the experiment.

The mean number of juveniles in the control group was 184.75 per replicate. The number of juveniles and as well as the percentage mean of juveniles was not statistically significant compared to the control group at the whole tested concentration range of 62.5 - 1000 mg/kg soil dry weight.

The EC50, based on the reproduction rate (juvenile numbers) of Eisenia fetida was determined to be higher than 1000 mg/kg soil dry weight, the highest tested concentration rate in the study.

There was an increase in earthworm biomass in the untreated control (3.49 %) and in all test item treated group. The biomass development was not statistically significantly inhibited at the whole tested concentration range of 62.5 - 1000 mg/kg soil dry weight (Dunnett’s Test, α=0.05).

 

CONCLUSION

The validity criteria of the study are met.

The test item LOWINOX® 44B25 has no adverse effect on the reproduction of earthworms (Eisenia fetida).