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Ecotoxicological information

Toxicity to terrestrial plants

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Administrative data

Endpoint:
toxicity to terrestrial plants: long-term
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
14 June 2000 - 5 July 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
other: study report - endpoints updated from author provided data
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: American Society for Testing and Materials (ASTM) method Standard Guide for Conducting Terrestrial Plant Toxicity Tests (E 1963-98)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): CoCl2. 6H2O
- Source: Fisher Scientific, 999 Veterans Memorial Drive, Houston, Texas
- Other: not reported
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Soil samples were analyzed for test substances using U.S. EPA contract laboratory program
(CLP) methods (U.S. EPA, 1999; 2000). The CLP contract required detection limits for the
analytical teclmiques were specified (Table 1.7). The elements cobalt, nickel, and selenium were
determined by inductively coupled plasma-atomic emission spectrometry (ICP-AES) using the
analytical protocol for ICP-AES analyses described in the CLP Statement of Work (SOW)
ILM 04.0, U.S. EPA method 200.7 CLP-M (U.S. EPA, 2000).

Soil samples of 100 g were placed in certified clean glass sample bottles, packed in ice and
shipped to Mitkem Corporation in Warwick, Rhode Island, for chemical analysis. Teflon scoops
were used to mix soils thoroughly before sampling and to collect soil samples. The scoops were
washed and decontaminated between mixing batches of soils as specified in ASTM method E
1963-98 (ASTM, 1999).

Test substrate

Vehicle:
not specified
Details on preparation and application of test substrate:
Two soils were defined for the project (Table 1.1). One was a synthetic soil consisting of sand,
10% kaolinite, and 5% finely ground peat moss with an adjusted pH fi'om 6.0 to 6.5. This
synthetic soil mixture content was based on recommendations from the U.S. EPA Eco-SSL Tsak
Group 4. The second soil \I,'as collected from a representative field location such that the soil had
a pH from 4.8 to 5.5, contained organic matter less than 2%, and had a correspondingly low clay
content. Soils were mixed and prepared according to the ASTM method.

A batch of each soil test material was prepared for each concentration ofthe different test
substances. These batches were allowed to age for four weeks prior to use. Soils were
moistened weekly to wetting and drying cycles important to simulate weathering or aging
processes known to temper bioavailability of substances added to soils.2 General instructions for
introducing the test substance into the test material are contained in ASTM method E1963-98
(ASTM, 1999).

Test organisms

Species:
other: Medicago sativa
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Alfalfa
- Plant family: Fabaceae

Study design

Test type:
other: Seedling emergence, growth, and survival
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
14 d
Post exposure observation period:
The effects of cobalt were evident by Day 7 post emergence; plants in the higher treatments \vere
stunted and exhibited chlorosis. As the duration of exposure increased, the separation of
treatments by size and shoot appearance also increased.

Test conditions

Test temperature:
22 degrees celcius
pH:
5.01
Moisture:
not reported
Details on test conditions:
TEST SYSTEM
- Testing facility:
- Test container (type, material, size):
- Amount of soil:
A total of 88 pots was prepared for each species in the definitive test (10 concentrations with
3-6 replicates x 2 soils).
- Mixing: Aliquots of the high concentration batch were mixed with clean soil to make
the lower concentration batches. These were first mixed by hand in plastic bags and then placed
into a cement mixer for 15 minutes. The batches were again mixed by hand and retumed to tbe
cement mixer for another 15 minutes of mixing. After each soil was mixed, it was stored at
ambient temperature for four weeks. The aging process also included a wetting and drying
process in which the soils were hydrated with deionized water and the bags left open to allow the
soil to dry.
Plant

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
a modified artificial soil mixture (artificial sloil) or a field-collected riverine soil from the Willamette Valley in Oregon (native soil)

NUTRIENT MEDIUM (if used)
- Description:

GROWTH CONDITIONS
16: 8 (light: dark) photoperiod was used.
Lighting was provided by Westinghouse Real Lite™, 48-inch fluorescent, 40 watt, f'ull spectrum
bulbs (Domestic Code F40TI2/FS).

ACCLIMATION PERIOD: not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

- Phytotoxicity rating system (if used): The results of the qualitative and quantitative observations were synthesized into a conunon
metric referred to as a "phytotoxic score." Measurements that were both statistically significant
and <90% of the corresponding value for the control (0 mg/kg Co, T-01) were assigned
categorical ranks. These were then totaled for all endpoints for a given species-soil combination.
A higher score indicates a greater magnitude of phytotoxicity.

VEHICLE CONTROL PERFORMED: no

TEST CONCENTRATIONS
The phytotoxic response to cobalt appeared to increase gradually over a relatively broad
concentration range. Consequently, the optimal concentration range to select for the definitive
tests was unceliain. For some species-endpoints, it appeared as if the threshold concentration
might be near 10 mg/kg, whereas other species-endpoints appeared to exhibit tolerance at
100 mg/kg or greater. Therefore, treatments \vere set bet\veen 4.0 and ~350 mg/kg for the
definitive tests.
Nominal and measured concentrations:
See table 2.11 below
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Species:
other: Medicago sativa
Duration:
14 d
Dose descriptor:
EC10
Effect conc.:
2.9 mg/kg soil dw
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: added Co in artificial soil
Basis for effect:
other: shoot dry weight
Remarks on result:
other: 95% CL: 1.0-8.1
Species:
other: Medicago sativa
Duration:
14 d
Dose descriptor:
EC10
Effect conc.:
6.6 mg/kg soil dw
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: added Co in artificial soil
Basis for effect:
other: shoot length
Remarks on result:
other: 95% CL: 4.5-9.7
Species:
other: Medicago sativa
Duration:
14 d
Dose descriptor:
EC10
Effect conc.:
5 mg/kg soil dw
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: added Co in artificial soil
Basis for effect:
other: root length
Remarks on result:
other: 95% CL: 2.5-10.1
Species:
other: Medicago sativa
Duration:
14 d
Dose descriptor:
EC10
Effect conc.:
8.6 mg/kg soil dw
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: added Co in artificial soil
Basis for effect:
other: root dry weight
Remarks on result:
other: 95% CL: 3.0-24.8
Species:
other: Medicago sativa
Duration:
14 d
Dose descriptor:
EC10
Effect conc.:
3.2 mg/kg soil dw
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: added Co in artificial soil
Basis for effect:
other: total dry weight
Remarks on result:
other: 95% CL: 1.2-8.4
Details on results:
All three species exhibited a strong phytotoxic response to cobalt. Many of the endpoints would
serve to illustrate the response; however, the most integrative response is total dry weight of the
plants (obtained by summing shoot and root dry weight measurements). At the lower
concentrations, there was a slight stimulation in plant growth, typical of an hormesis response
(Table 2.14). After treatment T-04 (12.3 mg/kg nominal concentration), plants exhibited a
steady decrease in growth. The highest concentration eliminated alfalfa.
The various endpoints were regressed against the log of the measured cobalt concentrations for
each treatment. In mailY instances linear regressions were significant, but a substantially better
fit was obtained using binomial regressions. Individual plots of each species-endpoint are
presented in the appendices. Plant endpoints showed strong concentration-dependent effects,
although virtually all endpoints exhibited characteristic hormesis response curves.
Consequently, better description of the concentration-response relationships generally were
obtained using binomial regressions where the endpoint values were regressed against the log
measured concentration of cobalt. Squared regression coefficients (R2) were consistently >0.6,
and many were >0.85 for all species-endpoints.
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
Dose-response curves of Kapustka et al. were refitted based on the raw data, with a log-logisctic (sigmoidal) model by minimising unweighted squared residuals sum (maximum likelihood).

Any other information on results incl. tables

Original results reported by Kapustka et al. can be found in the table below. Linear and binomial equations were used to calculate the EC20 values for each species-soil endpoint combination. The quadratic equation was used to solve for the concentration.

According to REACH guidance Chapter R.10 (Table R.10 -1) EC10 values are used preferentially for PNEC derivation, therefore EC20 values are only presented as supporting information. EC10 values used for effects assessment were derived from log-logistic dose response curves fitted on the raw data.

  Medicago sativa  14d   EC20 62.5  mg/kg soil dw   meas. (geom. mean)   total Co in artificial soil  seedling emergence
  Medicago sativa  14d    EC20  15.8  mg/kg soil dw   meas. (geom. mean)  total Co in artificial soil   shoot height
  Medicago sativa  14d    EC20 9.7   mg/kg soil dw  meas. (geom. mean)    total Co in artificial soil  root length
 Medicago sativa   14d    EC20  6.6  mg/kg soil dw   meas. (geom. mean)   total Co in artificial soil  shoot dry weight
  Medicago sativa  14d    EC20  6.1  mg/kg soil dw   meas. (geom. mean)  total Co in artificial soil   shoot dry weight/plant
  Medicago sativa  14d    EC20   5.5 mg/kg soil dw   meas. (geom. mean)   total Co in artificial soil  root dry weight
  Medicago sativa  14d   EC20  4.7  mg/kg soil dw   meas. (geom. mean)   total Co in artificial soil  root dry weight/plant
  Medicago sativa  14d    EC20   6.3 mg/kg soil dw   meas. (geom. mean)   total Co in artificial soil  total dry weight
 Medicago sativa  14d  EC20  5.8 mg/kg soil dw  meas. (geom.mean)  total Co in artificial soil  total dry weight/plant
 Medicago sativa  14 d  EC20  0.6 mg/kg soil dw  meas. (geom.mean)  total Co in artificial soil  nodule number

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The EC10 values used for PNEC derivation ranges from 2.9 to 8.6 mg added Co/kg soil.