Registration Dossier

Administrative data

Endpoint:
toxicity to terrestrial plants: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 April 2002 to 21 August 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 208 (Terrestrial Plants Test: Seedling Emergence and Seedling Growth Test)
Deviations:
yes
Qualifier:
according to
Guideline:
EPA OPPTS 850.4100 (Terrestrial Plant Toxicity Tier I (seedling emergence))
Deviations:
yes
Qualifier:
according to
Guideline:
EPA OPPTS 850.4225 (Seedling Emergence Tier II)
Deviations:
yes
Principles of method if other than guideline:
Sample analysis was conducted by LC/MS rather than HPLC. The analytical method was not validated prior to its use in the study, and was not added to the protocol by amendment. Samples were held in excess of one week between collection and analysis. It is considered that these deviations had no affect of the result of the study.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
The test substance was a composite of samples received from three manufacturers: Great Lakes Chemical Corporation (lot/batch CD-75P), Eurobrom BV (lot/batch 010328) and Albemarle Corporation (lot/batch 33449-15X). The composite material was analysed by Albemarle and considered to be homogenous.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations:
40, 105, 276, 725, 1904 and 5000 mg HBCD/kg dry soil

- Sampling method:
On the day of test soil preparation (May 8, 2002), three soil samples were collected from the 40, 105, 276, 725, 1904, and 5000 mg HBCD/kg treatment groups to verify the test concentrations and determine the homogeneity of the test substance in the carrier (soil). One sample was collected from the control group.

- Sample storage conditions before analysis:
Samples were stored at ambient room conditions until analysis was begun on May 9, 2002.

Test substrate

Vehicle:
no
Details on preparation and application of test substrate:
- Test soil:
The soil used for the test represented a loam soil, and was composed of kaolinite clay, industrial quartz sand, and peat mixed in a 4:50:2 ratio (w:w:w). Crushed limestone was added to buffer the pH of the soil, and a slow-release fertilizer was added to provide nutrients essential for plant growth. A sample of soil representative of that used in this study was sent to Agvise Laboratories, Inc., in Northwood, North Dakota, for analysis of the particle size distribution and organic matter content of the soil. The soil was determined to consist of 53% sand, 30% silt, and 17% clay, with an organic matter content of 1.9%, and a soil pH of 7.5.

- Method of mixing into soil:
The test soil was prepared by mixing HBCD into bulk test soil with a measured soil moisture of 14%. Test substance for treatment groups 40, 105, 276, 725, 1904, and 5000 mg HBCD/kg was prepared by weighing five known weights (2.1, 5.4, 14.2, 37.3, 98.1, and 257.5 g) of HBCD. Approximately 60 kg of bulk soil was measured into a soil mixer. Approximately 500 g of bulk soil was removed and added to a beaker containing test substance. The contents of the beaker were mixed by stirring and then evenly distributed across the top of the soil remaining in the mixer. The contents of the beaker and bulk soil were then mixed for ten minutes in order to prepare the test soil for each treatment group. Soils were mixed from lowest to highest concentration to avoid cross-contamination. The negative control soil was prepared in the same manner as the other test groups, but no test substance was added. At the completion of mixing, the test soils were sampled to provide material for analytical confirmation of the test concentrations. Analytical samples were stored at ambient room conditions after their collection until they were analyzed.

- Controls:
Untreated substate control.

Test organisms

open allclose all
Species:
Zea mays
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name:
Corn

- Plant family:
Poaceae

- Variety:
Mandan Bride

- Source of seed:
Johnny's Selected Seeds, Albion, ME, USA.

These species were chosen because they represent ecologically important families, and are readily cultivated test organisms that are widely used in research. Seeds were selected from a single size class within each species in order to reduce the potential for bias from differing seed sizes. Seeds used in this study were not treated with fungicides, insecticides or repellents prior to test initiation.
Species:
Allium cepa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name:
Onion

- Plant family:
Alliaceae

- Variety:
Texas Grano

- Source of seed:
Territorial Seed Co., Cottage Grove, OR, USA

These species were chosen because they represent ecologically important families, and are readily cultivated test organisms that are widely used in research. Seeds were selected from a single size class within each species in order to reduce the potential for bias from differing seed sizes. Seeds used in this study were not treated with fungicides, insecticides or repellents prior to test initiation.
Species:
Lolium perenne
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name:
Ryegrass

- Plant family:
Poaceae

- Variety:
Manhattan 3

- Source of seed:
Meyer Seed Co., Baltimore, MD, USA

These species were chosen because they represent ecologically important families, and are readily cultivated test organisms that are widely used in research. Seeds were selected from a single size class within each species in order to reduce the potential for bias from differing seed sizes. Seeds used in this study were not treated with fungicides, insecticides or repellents prior to test initiation.
Species:
Cucumis sativus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name:
Cucumber

- Plant family:
Cucurbitaceae

- Variety:
Straight Eight

- Source of seed:
Meyer Seed Co., Baltimore, MD, USA

These species were chosen because they represent ecologically important families, and are readily cultivated test organisms that are widely used in research. Seeds were selected from a single size class within each species in order to reduce the potential for bias from differing seed sizes. Seeds used in this study were not treated with fungicides, insecticides or repellents prior to test initiation.
Species:
Glycine max (G. soja)
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name:
Soybean

- Plant family:
Fabaceae

- Variety:
Green Envy

- Source of seed:
Johnny's Selected Seeds, Albion, ME, USA

These species were chosen because they represent ecologically important families, and are readily cultivated test organisms that are widely used in research. Seeds were selected from a single size class within each species in order to reduce the potential for bias from differing seed sizes. Seeds used in this study were not treated with fungicides, insecticides or repellents prior to test initiation.
Species:
Lycopersicon esculentum
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name:
Tomato

- Plant family:
Solanaceae

- Variety:
Rutgers

- Source of seed:
Meyer Seed Co., Baltimore, MD, USA

These species were chosen because they represent ecologically important families, and are readily cultivated test organisms that are widely used in research. Seeds were selected from a single size class within each species in order to reduce the potential for bias from differing seed sizes. Seeds used in this study were not treated with fungicides, insecticides or repellents prior to test initiation.

Study design

Test type:
seedling emergence toxicity test
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
none

Test conditions

Test temperature:
18-34.7 deg C
pH:
7.5
Moisture:
Water lost through transpiration and evaporation was replaced by subirrigation with well water from the greenhouse facility. Seedlings were subirrigated to minimize the potential for the leaching of the test substance through the soil. Subirrigation trays were filled to a predetermined depth to help standardize the amount of water delivered to each tray.
Details on test conditions:
TEST SYSTEM
- Testing facility:
Wildlife International, Ltd. greenhouse facility in Easton, Maryland.

- Test container (type, material, size):
plastic pots, approximately 16 cm in diameter and 12 cm deep

- Amount of soil:
not reported

- Method of seeding:
A template was used to gently compact the soil and leave ten uniform holes for planting. One indiscriminately selected seed was then planted in each hole, for a total of ten seeds in each pot. Holes were then closed by slightly depressing the soil surface.

- No. of seeds per container:
10

- No. of plants (retained after thinning):
not applicable

- No. of replicates per treatment group:
4

- No. of replicates per control:
4

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
The soil used for the test represented a loam soil, and was composed of kaolinite clay, industrial quartz sand, and peat mixed in a 4:50:2 ratio (w:w:w). Crushed limestone was added to buffer the pH of the soil, and a slow-release fertilizer was added to provide nutrients essential for plant growth. A sample of soil representative of that used in this study was sent to Agvise Laboratories, Inc., in Northwood, North Dakota, for analysis of the particle size distribution and organic matter content of the soil. The soil was determined to consist of 53% sand, 30% silt, and 17% clay, with an organic matter content of 1.9%, and a soil pH of 7.5. A copy of the complete report from Agvise Laboratories, Inc. was filed in the archives at Wildlife International, Ltd. along with the raw data for this study.

- Storage (condition, duration):
not reported

GROWTH CONDITIONS
- Photoperiod:
14 hours light/10 hours dark

- Light source:
Natural (sunlight), artificial lighting (high pressure sodium) was used to supplement natural sunlight in order to provide a uniform 14-hour photoperiod.

- Light intensity and quality:
not reported

- Day/night temperatures:
not reported

- Relative humidity (%):
19-81%

- Wind velocity:
not applicable

- Air exchange rate:
not applicable

- Watering regime and schedules:
Water lost through transpiration and evaporation was replaced by subirrigation with well water from the greenhouse facility. Seedlings were subirrigated to minimize the potential for the leaching of the test substance through the soil. Subirrigation trays were filled to a predetermined depth to help standardize the amount of water delivered to each tray.

- Interval of applications:
as necessary, Dates of watering: 08/05/02, 09/05/02, 13/05/02, 17/05/02, 20/05/02, 22/05/02, 24/05/02, 26/05/02, 28/05/02

ACCLIMATION PERIOD:
not applicable.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
seedling emergence and health on day 21.

- Seedling Condition Rating System:
The rating system below was used to help evaluate the health of seedlings on day 21. Assigned scores by treatment group are reported on the following pages.

Rating: Category: Description:
0 No Effect No noticeable effect
10 Slight Effect Effect barely noticeable
20 Slight Effect Some effect, not apparently detrimental
30 Slight Effect Effect more pronounced, not obviously detrimental
40 Moderate Effect Effect moderate, plants appear able to recover
50 Moderate Effect More lasting effect, recovery somewhat doubtful
60 Moderate Effect Lasting effect, recovery doubtful
70 Severe Effect Heavy injury, loss of individual leaves
80 Severe Effect Plant nearly destroyed, a few surviving leaves
90 Severe Effect Occasional surviving leaves
100 Complete Effect Death of entire plant

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
40, 105, 276, 725, 1904, and 5000 mg HBCD/kg

- Justification for using less concentrations than requested by guideline:
not applicable

- Range finding study:
not applicable
Nominal and measured concentrations:
Nominal: 40, 105, 276, 725, 1904 and 5000 mg HBCD/kg
Measured: 31.3, 97.8, 297, 764, 2230 and 6200 mg HBCD/kg
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Species:
other: All test species
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
5 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks on result:
other: no effects observed at any test concentration on any test species.
Species:
other: All test species
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
5 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
phytotoxicity
Remarks on result:
other: no effects observed at any test concentration on any test species.
Species:
other: All test species
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
6 200 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks on result:
other: no effects observed at any test concentration on any test species.
Species:
other: All test species
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
6 200 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
phytotoxicity
Remarks on result:
other: no effects observed at any test concentration on any test species.
Details on results:
The results of the test are summarized for each species in Tables 2 through 7 (attached). Complete results are presented by species in Appendices 6 through 11 (attached). There were no apparent effects on any endpoint for any of the six species tested. Statistical analyses indicated that there were no significant differences (Dunnett's test, p >0.05) between the control and treatment group mean emergence, survival, height, or weight for corn, cucumber, ryegrass, soybean and tomato. On day 21, onion showed a statistically significant difference (Dunnett's test, p <0.05) between the control and the 276 mg HBCD/kg treatment group mean survival. This significant difference was not considered dose-responsive, and not attributed to treatment, as no statistical differences were noted at higher concentrations tested (Dunnett's test, p >0.05). There were no statistically significant differences (Dunnett's test, p >0.05) between the control and treatment group mean emergence, height or weight for onion. Additionally, there were no signs of treatment-related phytotoxicity observed on seedlings of any species at any test concentration.
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
Statistical analyses were used to aid in the evaluation of effects of test substance application on seedling emergence, survival, mean shoot weight, and seedling height. These variables were defined for statistical analysis as follows:

- Seedling Emergence:
The number of emerged seedlings per ten planted seeds in each pot.

- Survival:
The number of emerged seedlings in each pot that were living at test termination per ten planted seeds.

- Mean Shoot Weight:
The average dry shoot weight of living emerged seedlings in each pot.

- Height:
The average height of living emerged seedlings in each pot.

Test data were evaluated to determine the no-observed-effect-concentration (NOEC) and lowest-observable-effect-concentration (LOEC) for condition and growth. The NOEC is defined as the maximum test substance concentration that shows no adverse phytotoxic effects and below which no phytotoxic effects are manifested. The LOEC is defined as the lowest test substance concentration used in the study that shows an adverse effect on a variable of interest. Mean seedling emergence, survival, weight, and height of the control and treatment groups were compared with Dunnett's t-test, using the DUNNETT option of the GLM (general linear model) procedure of SAS version 8 (5). Significance was determined at the level of 0.05 (p<0.05). Dunnett's test was used to aid in establishing the NOEC by determining which treatment groups differed significantly from the control group.

Applicant's summary and conclusion

Validity criteria fulfilled:
not specified
Conclusions:
No effects from soil incorporation of HBCD were observed on seedling emergence, survival, or growth for any of the six plant species tested. The NOEC for emergence and growth of all seedlings in this study was determined to be 5000 mg/kg (nominal).
Executive summary:

STUDY TITLE: Hexabromocyclododecane (HBCD): A Toxicity Test to Determine theEffects of the Test Substance on Seedling Emergence of Six Species ofPlants

GUIDELINES:     OECD Guideline for Testing of Chemicals, Proposal for Revision of Guideline 208: Terrestrial Non-Target Plant Tests

OPPTS 850.4100 (Public Draft)

OPPTS 850.4225 (Public Draft)

 

NOMINAL TEST LEVELS: 0 (Negative Control), 40, 105, 276, 725, 1904, 5000 mg HBCD/kg dry soil

MEAN MEASURED TEST LEVELS: 0 (Negative Control), 31.3, 97.8, 297, 764, 2230, and 6200 mg HBCD/kg dry soil

 

TEST DATES:               

STUDY INITIATION:                                   April 18, 2002

Experimental Start (OECD):                              May 8, 2002

Experimental Start (EPA):                                May 8, 2002

Experimental Termination:                                 June 3, 2002

STUDY COMPLETION:                                 August 21, 2002

 

LENGTH OF TEST: 21 days

 

TEST SPECIES: Corn (Zea mays),Cucumber (Cucumis sativa),Onion (Allium cepa),Ryegrass (Lolium perenne),Soybean (Glycine max),Tomato (Lycopersicon esculentum)

RESULTS: The soil incorporation of Hexabromocyclodododecane (HBCD) caused no effects onemergence, survival, or growth for any of the six plant species tested. Therefore, the highest nominal soil concentration tested, 5000 mg HBCD/kg dry soil, was considered to be the NOEC for these test species.