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Toxicity to soil macroorganisms except arthropods

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Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 19.04.2017
- Expiration date of the lot/batch: 19 Apr 2022
- Purity test date: 100% (as UVCB)
Analytical monitoring:
no
Vehicle:
yes
Details on preparation and application of test substrate:
- Method of mixing into soil (if used): The test substance C9-Aldoxime is liquid and poorly soluble in water. As the test substance is soluble in acetone, a stock solution in acetone was prepared. The amount of 25000.4 mg test substance was weighed into a 50 mL volumetric flask. The test substance was dissolved by addition of acetone as solvent. The solution was filled up to the mark afterwards. The test substance was completely dissolved. The solution was clear and yellowish.
Aliquots of the stock solution with required amounts of test substance for each scheduled test concentration were carefully mixed with quartz sand. The organic solvent was removed by evaporation and subsequently blended with prepared test substrate. Thereafter, the water content of the soil was adjusted to about 50% WHCmax using deionized water and was mixed once again. The test mixtures were distributed to the test units.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: earthworm
- Source: in-house breeding
- Age at test initiation (mean and range, SD): 9 months
- Weight at test initiation (mean and range, SD): 300 - 600 mg

ACCLIMATION
- Acclimation period/conditions: Before starting the test, the worms selected were acclimatized in the test substrate in a plastic box under test conditions through overnight under permanent light and without feeding. For this, a plastic box was filled up with approx. 750 g test substrate (Dw) and added with a sufficient number of adult worms that were sorted out from the breeding boxes.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
28 d
Test temperature:
20.5 ° C – 21.8 °C (exception on 22nd – 27th days of exposure with ≤ 0.5 °C increase in measurement due to technical reasons; with 22.5 ° C on 22nd day and 22.4 °C on 27th day of exposure)
pH:
5.7 - 5.9 (start of exposure)
6.8 - 7.0 (end of exposure)
Moisture:
50 ±10 % WHCmax (mean value)
Details on test conditions:
TEST SYSTEM
- Test container (material, size): Plastic dishes with a volume of about 1.0 L, dimensions 11×15.5×6 [cm] with transparent and punctured lid
- 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: 8

OTHER TEST CONDITIONS
- Photoperiod: 16h light: 8h dark
- light intensity: 605 Lux, (mean of 5 single measurements: 769, 641, 429, 673 and 513 lux)

EFFECT PARAMETERS MEASURED: Mortality, weight and reproductive output was recorded throughout the test period of 28 days.

VEHICLE CONTROL PERFORMED: yes
Nominal and measured concentrations:
Nominal: 62.5, 125, 250, 500 and 1000 mg/kg
Reference substance (positive control):
yes
Remarks:
Carbendazim (BAS 346 F)
Key result
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
94 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
125 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
125 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: biomass change
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
Details on results:
- Mortality at end of exposure period: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: 28d-EC50 = 1.19 mg/kg dw
Validity criteria fulfilled:
yes
Conclusions:
The 28d-EC10 was determined to be 94 mg/kg dw.
Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Acceptable, well documented and peer reviewed publication which meets basic scientific. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Justification for type of information:
Analogue justification attached.
Qualifier:
no guideline available
Principles of method if other than guideline:
Study on the effect of increasing NP concentrations in soil microcosms containing a simplified soil community over a test period of 112 days.
GLP compliance:
not specified
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
- Method of mixing into soil (if used): Due to its high hydrophobicity, different solutions of 4-nonylphenol in acetone (95%, Panreac, Barcelona, Spain) were prepared in order to achieve different soil concentrations (0, 10, 30, 90, and 270 mg/kg of soil). For each concentration, the soil was hydrated with a fixed volume of acetone solution containing the appropriate quantity of NP (150 mL of solution per kilogram of soil), a rate which permitted proper spiking of the chemical solution with soil. An equal acetone solution volume was applied to all the test concentrations. Pure acetone was added for the controls. The acetone was then left to evaporate for 24 h in a fume hood. For each concentration prepared, a small portion of the untreated soil (20%) was put aside to be used as microbial inoculums prior to the addition of the acetone solution. Once the acetone had evaporated and just before the beginning of the experiment, this soil portion was mixed with the polluted soil in order to reinoculate it with the original microbial soil community. Just before the construction of the microcosms, the soil batches were hydrated to 25% (corresponding to 45% of their water holding capacity). This moisture corresponded to the maximum water content that allowed this soil to have a crumbly structure, due to its loamy nature.

Test organisms (species):
other: simplified soil community (microorganisms, seedling of Avena sativa, Porcellionides sexfasciatus, Enchytraeus crypticus, Folsomia candida, Ceratophysella denticulata, Proisotoma minuta, Hypoaspis aculeifer)
Animal group:
other: microorganisms, plants, nematods, collembolans, isopods, mites
Details on test organisms:
The microcosms consisted of an assemblage of cultured organisms which mimicked a soil invertebrate food web. Decomposers were represented as the natural microbial community, since spiked soil was reinoculated with unspiked soil just before the start of the experiment, as described above. As primary producers, only one seedling of common oats (Avena sativa) was used per microcosm, due to the small size of the containers and in order to prevent excessive root growth in the soil. As detritivores (consumers), four species belonging to three different functional groups were used. Isopods of the species Porcellionides sexfasciatus (Crustacea, Isopoda, Oniscidea) represented the macrofauna living on the soil surface. The individuals came from a laboratory culture initially started with wild individuals collected in southern Portugal (São Domingos, Alentejo, Portugal). As representatives of the mesofauna, three collembolan species and an enchytraeid were introduced into the microcosms. The collembolan species used were F. fimetaria (Collembola, Isotomidae), Proisotoma minuta (Collembola, Isotomidae), and Ceratophysella (Hypogastrura) denticulata (Collembola, Poduridae). All these species were cultured in the laboratory for at least 3 years and were obtained, respectively, from the National
Environmental Research Institute (Silkeborg, Denmark), from an earthworm culture in our laboratory, and from the collembolan “bloom” of an agricultural soil in central Catalonia (NE Spain). Enchytraeus crypticus (Oligochaeta, Enchytraeidae) were also bred in our laboratory and used as a representative of soil enchytraeids. Finally, as a representative of soil predators, mites of the species Hypoaspis aculeifer (Acari, Gamasidae), provided by ECT Oekotoxikologie GmbH (Flörsheim, Germany), were introduced into the microcosms. All the soil species selected are able to carry out sexual reproduction, an important feature for the chemical assessed in this study, as it is alleged to have estrogenic effects as its main toxicity mechanism.
Study type:
semi-field study
Substrate type:
natural soil
Limit test:
no
Total exposure duration:
112 d
Test temperature:
20 ± 0.1 °C
pH:
8.3
Moisture:
70% relative humidity
Details on test conditions:
TEST SYSTEM
- Test container (material, size): plastic transparent polyethylene terephthalate bottles (16 cm in height and 8.5 cm in diameter) with open tops.
- Amount of soil or substrate: Each container was filled with 2 cm of quartz sand which, together with several holes in the basis of the container, allowed proper drainage of any excess water. Each container was then filled with 300 g of wet soil (previously contaminated), which produced a soil layer of around 10 cm.
- No. of organisms per container (treatment): The soil was then sown with five common oat seeds, and the NP was left to stabilize for 24 h. The oat seeds germinated approximately 4 days later. The day after setting up the microcosms (day 1), three adult isopods (P. sexfasciatus) per microcosm were added, one ma and two nonpregnant females. A single alder leaf (Alnus glutinosa) was also added to each microcosm, all with a similar weight. The leaves had previously been dried at 80°C for 24 h. The leaves were used as a food source by the isopods, thus minimizing the consumption of oat seedlings. On the same day, 15 adult individuals of P. minuta and 15 adult individuals of C. denticulata were added to each microcosm. The individuals came from mixed-age laboratory cultures and were selected randomly in order to provide similar sex ratios and adult age classes to all the microcosms. On day 2, ten adult enchytraeids (clearly identified by the clitellum) and 15 adult collembolans (F. fimetaria) were added to each microcosm. On day 6, six adult mites (H. aculeifer), three males and three females, were added to each microcosm. The reason for the delay in the addition of the mites was to allow the collembolans and the enchytraeids to reproduce and ensure their performance in the microcosms but also to minimize any starvation period for the mites. On the same day, all the oat seedlings in each microcosm were removed except one, which was the most vigorous plant.
- No. of replicates per treatment group: 15
- No. of replicates per control: 15

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
The soil used in the microcosms was the upper layer (20 cm) of a loamy natural soil collected from an experimental agricultural soil within the campus of the Autonomous University of Barcelona (Cerdanyola del Vallès, Spain). The soil had formerly been used for grain production and had been free of pesticides for at least 5 years. After collection, the soil was air-dried for a week and sieved (5 mm). To remove the natural invertebrate communities that could influence the results, the soil was defaunated by two consecutive freezing–thawing cycles, each consisting in placing soil at −20°C for 4 days followed by a period of 4 days at 20°C.
- % sand: 36.4
- % silt: 44.9
- % clay: 18.7
- Organic carbon (%): 2.63
- CEC: 13.9 meq/100g

OTHER TEST CONDITIONS
- Photoperiod: 16h light: 8h dark

EFFECT PARAMETERS MEASURED: Abundances of the different species observed was recorded after 28, 56 and 112 days exposure time.
Nominal and measured concentrations:
Nominal: 10, 30, 90 and 270 mg/kg
Reference substance (positive control):
no
Key result
Duration:
112 d
Dose descriptor:
NOEC
Effect conc.:
90 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: abundance
Remarks on result:
other: related to soil community
Details on results:
The results from this study indicate that concentrations above 90 mg NP kg–1 may cause changes in soil invertebrate communities. The maximum concentration tested (270 mg NP kg–1) changed the invertebrate community significantly, although a recovery was observed after 112 days.
Reported statistics and error estimates:
The effects of nonylphenol on the microcosm community over time, that is, the effects of pollution ruling out the community intrinsic temporal changes, were assessed by multivariate analysis, using principal response curves (PRC).
Validity criteria fulfilled:
not applicable
Conclusions:
A 112d-NOEC for a soil community is determined to be 90 mg/kg dw based on abundance in a semi-field study (microcosms).
Executive summary:

The effect of increasing 4-nonylphenol (NP) concentrations (0, 10, 30, 90, and 270 mg/kg) in soil microcosms containing a simplified soil community was investigated over three sampling dates (28, 56, and 112 days) using the principal response curves method. The soil community did not change significantly at concentrations below 90 mg/kg, which was selected as the nonobserved effect concentration (NOEC). The highest concentration (270 mg/kg) changed the community significantly after 28 and 56 days, but this effect disappeared after 112 days.

Description of key information

EC10(28d) = 94 mg/kg dw (nominal) for reproduction of Eisenia fetida (OECD 222)

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for soil macroorganisms:
94 mg/kg soil dw

Additional information

One study investigating the long-term toxicity of the test substance to soil macroorganisms is available. The study was performed according to OECD guideline 222 under GLP conditions using Eisenia fetida as test organism (BASF 2018). Acetone was used as solvent to a prepare stock solution of the test substance. The organic solvent was removed by evaporation and subsequently blended with prepared test substrate. Nominal concentrations between 62.5 and 1000 mg/kg dw were tested for effects on survival, weight and reproductive output throughout a test period of 28 days. No mortality was observed in the controls and any treatment. The 28d-NOEC value for reproduction was determined to be 125 mg/kg dw. The 28d-EC10 for reproduction is determined to be 94 mg/kg dw.

Additional data are available for Phenol, 4-nonyl-,branched, which is a secondary component and structurally similar to the main component of the substance. The only structural difference between the source substance and the target substance is the lack of an aldoxime group at the phenol ring of the molecule. The effect of increasing 4-nonylphenol (NP) concentrations (0, 10, 30, 90, and 270 mg/kg) in soil microcosms containing a simplified soil community was investigated over three sampling dates (28, 56, and 112 days) using the principal response curves method (Domene et al. 2010). The soil community did not change significantly at concentrations below 90 mg/kg, which was selected as the NOEC value. The highest concentration (270 mg/kg) changed the community significantly after 28 and 56 days, but this effect disappeared after 112 days.