Reaction mass of cobalt sulphide and nickel sulphide and trinickel disulphide

Toxicity to aquatic plants other than algae

Administrative data

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with minimal documentation on water quality parameters.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Deviations:

yes

Remarks:

:Test acceptability for OECD is 7-fold doubling while ASTM is 5-fold doubling. In order to keep Ni in solution during the exposure period the pH was lowered. Due to this change the 7-fold doubling was not achieved, although a >5-fold doubling was achieved

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Nickel sulphate

EC Number:

232-104-9

EC Name:

Nickel sulphate

Cas Number:

7786-81-4

Molecular formula:

H2O4S.Ni

IUPAC Name:

Nickel sulfate

Constituent 2

Reference substance name:

Hexahydrate

IUPAC Name:

Hexahydrate

Details on test material:

Source: Aldrich, Milwaukee, Wl, USA
Purity: 99.99%

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not relevant

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

water samples were taken for AAS analysis, DOC analysis, ICP analysis, pH, water hardness, and alkalinity. AAS and ICP samples
were acidified with concentrated nitric acid (Trace metal grade, Fisher, Pittsburg, PA, USA) and stored for later analysis.

Test solutions

Vehicle:

not specified

Test organisms

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Common name: Duckweed
- Strain: Not reported
- Source (laboratory, culture collection): Dr. Yamamoto, North Carolina State University
- Age of inoculum (at test initiation): Not reported
- Method of cultivation: Divided on a monthly basis in modified 20X APP growth
medium, under cool-white fluorescent lights at 12000 lux, 24-hour photoperiod, and 26
(+ 1) oC. Cultures were grown in 250 ml pyrex containers with Petri-dish lids,
maintaining sterile conditions, while allowing for gas exchange.


ACCLIMATION
- Acclimation period: Test plants were placed in control medium prior to test. Selection criteria for test
plants were health of the fronds, similarity of frond size, and having 2-3 fronds per plant.
The tests were performed in similar containers and under similar conditions as the
culture. Although the culture was not truly axenic, procedures were performed using
aeseptic techniques to keep bacterial contamination at a minimum and avoid algal
contamination.
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: Not reported

Study design

Test type:

other: static and static-renewal tests

Water media type:

freshwater

Total exposure duration:

7 d

Test conditions

Hardness:

Artificial water medium: 300 mg/L CaCO3
natural water: 40 mg/L CaCO3

Test temperature:

not reported

pH:

6.0 - 7.2

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Nominal and measured concentrations:

20X-APP Growth Medium— Static Test Nominal Concentrations: 0.125 - 4 mg Ni/L
PD113 Site Water— Static Test Nominal Concentrations: 0.0625 - 2 mg Ni/L
20X-APP Growth Medium— Static Renewal Test Nominal Concentrations: 0.0625 - 2 mg Ni/L

Details on test conditions:

20X-APP Growth Medium— Static Test
Medium was formulated according to the Lemna sp. Growth Inhibition Test guidelines (OECD 2003) for 20X APP growth medium;
however, the Na2EDTA.2H2O was omitted, and only 50 % of the NaHCO3 was added. Medium was brought to volume
with milli-q water, and the pH was adjusted to approximately 6 with 1 N HCl. To sterilize the medium, all equipment was
autoclaved at 121oC for 15 minutes and the medium was filtered using sterile 0.2 μm Supor-200 membrane filters (Pall Corp., Ann
Arbor, MI, USA). Nickel was added to treatment solutions for a total of 6 treatments and a control of the same nutrient solution with 4
replications each. 200 ml of treatment solution was added to autoclaved test containers and allowed to set for 24 hours.
Approximately 2 to 3 duckweed colonies, totaling 10 fronds, were axenically transferred to test containers, placed under test conditions
for 7 days. All containers were randomized on days 1, 3, 5, and 7. Frond count and dry weight measurements were taken
on day 7. Water samples were taken from each treatment on day 0 to determine total Ni, dissolved organic carbon (DOC), selected ion
concentrations, pH, water hardness, and alkalinity.

PD113 Site Water— Static Test
Water was collected from the Lynches River under the SC Hwy 9 Bridge on the border of Lancaster and Chesterfield Counties
in SC using the surface grab method. Water was filtered to remove large particles using a Whatman #1 qualitative filter
(Whatman International, England) and stored for 20 days at 4oC until use. PD113 water was adjusted to pH 6 with 1 N HCl.
To sterilize the water, all equipment was autoclaved at 121 deg C for 15 minutes and the water was filtered using sterile 0.2 μm filters.
No growth medium was added to the site water. Nickel was added to treatment solutions for a total of 6 treatments and a control solution
of filtered PD113 site water with 4 replications each. 200 ml of treatment solution was added to autoclaved test containers and allowed to
set for 24 hours. Approximately 2 to 3 duckweed colonies, totaling 10 fronds, were axenically transferred to test containers and placed
under test conditions for 7 days. All containers were randomized on daily after fronds were counted; and dry weight measurements were
taken on day 7. Water samples were taken from each treatment on day 0 for AAS analysis, DOC analysis, ICP analysis, pH, water hardness,
and alkalinity.

20X-APP Growth Medium— Static Renewal Test
As with the static test, medium was made up according to the Lemna sp. Growth Inhibition Test guidelines (OECD 2003) for 20X APP
growth medium; however the Na2EDTA.2H2O was omitted, and only 50 % of the NaHCO3 was added. Medium was brought to volume
with Milli-q water (18 meg-ohm). On Day 0, the pH was adjusted to approximately 6 with 1 N HCl. To sterilize the medium, all equipment
was autoclaved at 121oC for 15 minutes and the medium was filtered using sterile 0.2 μm filters. Nickel was added to treatment solutions
for a total of 6 treatments and a control of the same nutrient solution with 4 replications each. 100 m of treatment solution was added to
autoclaved test containers. The volume was changed from the previous bioassays because the plants would only be in the solution for 24 h.
Solution was not allowed to set for 24 hours and used immediately, however, because we wanted to use the solution before the pH raised
above 7, to minimize Ni complexation. Approximately 2 to 3 duckweed colonies, totaling 8 fronds, were axenically transferred to test containers
and randomly placed under test conditions. Fresh treatment solutions were made up daily, adjusted to a pH of approximately 5, and filtered
for sterilization. The solution was adjusted to pH 5 in order to keep the pH low for the 24 h between renewals. For the daily renewal, 80 % of the
test medium was siphoned from the container and replaced with fresh medium. Test containers were then randomly placed under test conditions
until the next renewal. Medium was replaced daily for a total of a 7 day exposure. Three containers with control medium but without plants were
treated similarly to test containers to measure pH fluctuations throughout the 7-day exposure. . Fronds were counted daily prior to renewal,
and dry weight measurements were taken on day 7. Water samples were taken from fresh medium daily for AAS analysis, DOC analysis,
ICP analysis, water hardness, and alkalinity.

EFFECT PARAMETERS MEASURED
Measurements Percent of control was used to report all endpoints. This is determined by dividing the individual measurements by the
average of the control. This allows for comparison between tests by normalizing all measurements to the control measurements.

Growth
The total number of fronds was counted either daily or at the beginning and end of the exposure depending on the test. Growth
(Frond count) is found by determining the difference between the total number of fronds at the end and the beginning of the test.
Dry weight was found by filtering the solution and duckweeds from each treatment container through a Whatman #1 qualitative filter,
rinsing the plants and filter with milli-q water, moving the plants to a pre-weighed aluminum weighing dish and drying the plants in an
oven at 90 deg C for 48 hours. The dried boat and plants were then weighed to determine ending dry weight. Four sets of 10 representative
fronds were collected from the mass culture used to start the test, dried, and weighed to estimate the beginning dry weight. Growth (Dry wt)
is found by determining the difference between the dry weight at the end of the test and an estimate of the dry weight at the beginning.

Growth Rate
Growth rate was determined by plotting the natural log of the daily frond count versus the hour of the test. The slope of the regression line
developed was determined for the growth rate in fronds/hour.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

ca. 0.19 other: mg Ni/L

Conc. based on:

other: 20XAPP Static Test

Basis for effect:

other: Growth (dry matter weight)

Effect concentrations 2

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

ca. 0.075 other: mg Ni/L

Conc. based on:

other: 20XAPP Statice Renwal Test

Basis for effect:

other: Growth (dry matter weight)

Effect concentrations 3

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

ca. 0.15 other: mg Ni/L

Conc. based on:

other: Site Water (PD113) Test

Basis for effect:

other: Growth (dry matter weight)

Effect concentrations 4

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

ca. 0.16 other: mg Ni/L

Conc. based on:

other: 20XAPP Static Test

Basis for effect:

other: Growth (frond count)

Effect concentrations 5

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

ca. 0.05 other: mg Ni/L

Conc. based on:

other: 20XAPP Static Renewal Test

Basis for effect:

other: Growth (frond count)

Effect concentrations 6

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

ca. 0.16 other: mg Ni/L

Conc. based on:

other: Site Water (PD113) Test

Basis for effect:

other: Growth (frond count)

Effect concentrations 7

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

ca. 0.07 other: mg Ni/L

Conc. based on:

other: 20XAPP Static Renewal Test

Basis for effect:

growth rate

Effect concentrations 8

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

ca. 0.19 other: mg Ni/L

Conc. based on:

other: Site Water (PD113) Test

Basis for effect:

growth rate

Effect concentrations 9

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

ca. 1.33 other: mg Ni/L

Conc. based on:

other: 20XAPP Static Test

Basis for effect:

other: Growth (dry matter weight)

Effect concentrations 10

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

ca. 0.2 other: mg Ni/L

Conc. based on:

other: 20XAPP Static Renewal test

Basis for effect:

other: Growth (dry matter weight)

Effect concentrations 11

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

ca. 0.3 other: mg Ni/L

Conc. based on:

other: Site Water (PD113) Test

Basis for effect:

other: Growth (dry matter weight)

Effect concentrations 12

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

ca. 0.75 other: mg Ni/L

Conc. based on:

other: 20XAPP Static Test

Basis for effect:

other: Growth (frond count)

Effect concentrations 13

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

ca. 0.15 other: mg Ni/L

Conc. based on:

other: 20XAPP Static Renewal Test

Basis for effect:

other: Growth (frond count)

Effect concentrations 14

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

ca. 0.3 other: mg Ni/L

Conc. based on:

other: Site Water (PD113) Test

Basis for effect:

other: Growth (frond count)

Effect concentrations 15

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

ca. 0.1 other: mg Ni/L

Conc. based on:

other: 20XAPP Static Renewal Test

Basis for effect:

growth rate

Effect concentrations 16

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

ca. 0.4 other: mg Ni/L

Conc. based on:

other: Site Water (PD113) Test

Basis for effect:

growth rate

Details on results:

The toxicity of nickel to L. gibba varied among the different conditions tested and the different endpoints measured.
For example, the EC50 values ranged from a low of 0.1 mg/L for growth rate in the static renewal test to 1.33 for growth as dry
weight in the static test. A comparison of the static versus static renewal test results reflects the difference in nickel bioavailablility as a
function of varying pH. For example, the static bioassays had an equilibrium pH of 7.2 while the static renewal bioassays began each 24hr
period at pH 6.0 and ended at approximately 7.2 to give a time-weighted average pH of about 6.5. In the lowest nickel concentrations at pH 6.0,
ionic nickel is approximately 84% of total dissolved nickel. At pH 6.5, ionic nickel drops to 81% of total dissolved nickel and at pH 7.2 it drops to
73% of total dissolved nickel. Measured values of Ni in all treatments were within 90% of nominal concentrations.

Reported statistics and error estimates:

A randomized block design was used for all tests. Plants were randomly assigned to treatment containers, and all containers were randomly
placed under lights throughout the test.
All Effective Concentrations (EC10, EC25, and EC50) were determined with by using a Linear Interpolation Method, ICp Version 2.0
((Norberg-King 1993). An Excel program developed within our laboratory using the EC’s, their standard deviations, and a student’s t-test
was used to determine differences between EC’s. No Observable Adverse Effect Concentrations (NOAEC) and Lowest Observable Adverse
Effect Concentrations (LOAEC) were determined by Dunnett’s Tests using SAS (SAS 1989).

Any other information on results incl. tables

Growth (dry weight and frond count) and growth rate were the measured endpoints at day 7 and generated EC₁₀values between 50 and 190 µg Ni/l. All effective concentrations were determined using linear interpolation method. The validity criterion, i.e. a 5 (ASTM, 2003) to 7 (OECD, 2003) fold doubling, was met in all the tests.

Reliability/relevance: Results from the static-renewal portion of the study were accepted. pH in the static test reached an equilibrium value of 7.2.  At this pH and at the test hardness of 300 mg/L CaCO₃, chemical speciation modeling indicated that precipitation of NiCO₃ was possible. Results from the static test were therefore rejected. The reliable/relevant EC₁₀ values varied between 50 and 80 µg Ni/l.

Applicant's summary and conclusion

Executive summary:

STUDY RATED BY AN INDEPENDENT REVIEWER.

ROBUST SUMMARY DEVELOPED BY AN INDEPENDENT REVIEWER.