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Administrative data

Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
long-term (30 d, 26 d post-hatch)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in accordance with a recognised guideline (draft at the time the study was performed) and to GLP. The validity criteria of the current guideline were fulfilled.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
- Draft version of November 1988
Deviations:
yes
Remarks:
: The study duration (30 d total, 26 d post-hatch) is slightly shorter than the current guideline recommendation of 30 d post-hatch for B. rerio.
Principles of method if other than guideline:
Not relevant.
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of chromium (+ 3) hydroxide sulfate and sodium sulfate
EC Number:
914-129-3
Cas Number:
12336-95-7
Molecular formula:
CrHO5S
IUPAC Name:
Reaction mass of chromium (+ 3) hydroxide sulfate and sodium sulfate
Details on test material:
- Name of test material (as cited in study report): 'basisches Chromsulfat'
- Molecular formula (if other than submission substance): Cr2(OH)2(SO4)2*Na2SO4
- Molecular weight (if other than submission substance): 472
- Physical state: Solid (powder).
- Analytical purity: 80% Cr2(OH)2(SO4)2*Na2SO4 plus ca. 20% water.
- Impurities (identity and concentrations): traces, identity not stated.
- Purity test date: not stated.
- Lot/batch No.: AC-P4/T18
- Expiration date of the lot/batch: not stated.
- Stability under test conditions: not stated.
- Storage condition of test material: not stated.
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:
Samples were taken from one of each pair of duplicate test vessels of the d 2, d14 and d 28 (fresh) and d 4, d 16 and d 30 (expired) media of the control groups and each of the 'basisches Chromsulfat' treatments, filtered and acidified prior to analysis to determine concentrations of dissolved Cr.
Samples were also taken from five concentrated dosing stock "solutions" containing 100 g 'basisches Chromsulfat'/L in distilled water and diluted prior to analysis to detemine total Cr.

- Sampling method: not stated. Test media samples are described as "supernatants", which implies mid-water samples without prior re-suspension of test material deposited on the base of the test vessels. Samples of freshly prepared media were taken after pH-adjustment.

- Sample storage conditions before analysis: not stated. Samples were generally filtered immediately after collection and acidified directly afterwards, with the exception of d 4 expired media samples which were not processed until 3 days after collection.

Test solutions

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 'basisches Chromsulfat' "dissolved" in distilled water in dosing stock solutions containing 20 g/200 mL (=100 g/L).
- Controls: unamended medium comprising groundwater fortified with mineral salts, also vehicle control consisting of the same medium amended with distilled water at a rate of 10 mL/L.
- Chemical name of vehicle: distilled water.
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 15 mL/1.5 L (= 10 mL/L) but lies outside the scope of the organic solvents usually understood in the context of vehicle or co-solvent (e.g. acetone, acetonitrile, DMSO etc) and the guideline limit on such substances does not apply.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): pH-adjusted media containing 'basisches Chromsulphat' at 3.2 - 100 mg/L described as "turbid" with undissolved test substance also on the base of the test vessels. At 1000 mg/L: no test substance in suspension, but deposits on base of test vessels.

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebra fish
- Species: Brachydanio rerio
- Source: commercial supplier, M.B. Ruijsbroek B.V., Maassluis, the Netherlands.


METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): Six adult females transferred singly to basins each containing 3 male fish already acclimatised to the conditions of confinement for 10 h. The eggs provided by the six fish were evenly distributed between all the test treatments., including the control.
- Method of collection of fertilised eggs: not stated.
- Subsequent handling of eggs: 80 eggs were placed in test vessels within 6 h of spawning and the stage of embryonic development (young blastula) at test initiation was verified by microscopic examination. After the initial 24 h of exposure, fertilised eggs were thinned to 35 per vessel.


POST-HATCH FEEDING
- Start date:
- Type/source of feed: initially with rotifers (Brachionus rubens) from a stock maintained at the test facility, supplemented from day 10 onwards with Artemia nauplii enriched with Selco.
- Amount given: ad libitum.
- Frequency of feeding: not stated.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
30 d
Remarks on exposure duration:
(26 d post-hatch)
Post exposure observation period:
Not relevant.

Test conditions

Hardness:
207-210 mg CaCO3/L.
Test temperature:
24.0 to 24.5 degrees C in all treatments.
pH:
0 mg 'basisches Chromsulfat'/L (groundwater control): pH 7.9 to 8.3 in all fresh media, pH 7.3 to 8.0 in expired media.
0 mg 'basisches Chromsulfat'/L (distilled water control): pH 7.9 to 8.3 in all fresh media, pH 7.3 to 8.2 in expired media.
3.2 mg 'basisches Chromsulfat'/L: pH 7.8 to 8.2 in fresh media before neutralisation, pH 7.9 to 8.3 in fresh media after neutralisation, pH 7.3 to 8.0 in expired media. .
10 mg 'basisches Chromsulfat'/L: pH 7.6 to 7.9 in fresh media before neutralisation, pH 7.9 to 8.3 in fresh media after neutralisation, pH 7.3 to 8.0 in expired media.
32 mg 'basisches Chromsulfat'/L: pH 7.1 to 7.5 in fresh media before neutralisation, pH 7.9 to 8.3 in fresh media after neutralisation, pH 7.3 to 8.0 in expired media.
100 mg 'basisches Chromsulfat'/L: pH 6.0 to 6.8 in fresh media before neutralisation, pH 7.9 to 8.3 in fresh media after neutralisation, pH 7.3 to 8.0 in expired media.
320 mg 'basisches Chromsulfat'/L: pH 5.3 to 5.7 in fresh media before neutralisation, pH 7.9 to 8.3 in fresh media after neutralisation, pH 7.3 to 8.0 in expired media.
1000 mg 'basisches Chromsulfat'/L: pH 4.6 to 5.0 in fresh media before neutralisation, pH 7.9 to 8.3 in fresh media after neutralisation, pH 7.6 to 8.0 in expired media
Dissolved oxygen:
8.1 to 9.3 mg O2/L in fresh media (all treatments).
5.5 to 8.3 mg O2/L in expired media (all treatments), excluding anomalously low values recorded in d 21 in samples left unaerated - with food detritus but without fish - for a long time after media renewal.
Salinity:
Not relevant.
Nominal and measured concentrations:
See analytical results table below.
Details on test conditions:
TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume):
- Test vessel: beakers.
- Type (delete if not applicable): covered with a clock glass.
- Material, size, fill volume: glass, 2 L capacity, containing 1.5 L medium. All test vessels contained a cylindrical perspex insert (10 cm diameter, x 16 cm tall) with a nylon mesh base to retain and support the eggs/larvae. These inserts were necessary, particularly at concentrations >/= 32 mg 'basisches Chromsulfat', to keep the test organisms out of the undissolved test substance.
- Aeration: yes.
- Renewal rate of test solution (frequency/flow rate): Media renewals at intervals of 2-3 days.
- No. of fertilized eggs/embryos per vessel:
- No. of vessels per concentration (replicates): two.
- No. of vessels per control (replicates): two.
- No. of vessels per vehicle control (replicates): two.
- Biomass loading rate:


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Groundwater obtained near Linschoten, the Netherlands, fortified with mineral salts (sodium: 1.09-1.20 mmol/L, potassium: 0.20-0.23 mmol/L, calcium: 1.35-1.37 mmol/L, magnesium 0.72-0.78 mmol/L, chloride: 2.59-2.64 mmol/L and sulphate: 0.67-0.72 mmol/L) , prepared in two 10,000 L batches for this study.
- Total organic carbon: 1.09-1.14 mg/L.
- Particulate matter: not stated.
- Metals: not stated.
- Pesticides: not stated.
- Chlorine: not stated.
- Alkalinity: not stated.
- Ca/Mg ratio: 1.76-1.88 (mol/mol)
- Conductivity: not stated.
- Salinity: not relevant.
- Culture medium different from test medium: not relevant.
- Intervals of water quality measurement: test initiation, thereafter every 2 to 3 days, coinciding with times of media renewal and at termination.


OTHER TEST CONDITIONS
- Adjustment of pH: yes.
- Photoperiod: 16 h.
- Light intensity: not stated.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : numbers of dead eggs/larvae, numbers of survivors, observations of swimming behaviour and of the presence of malformations were made at each medium renewal and at termination. Length and wet weight of individual survivors was determined at test termination and group-mean dry weights were determined after drying at 60 degrees C for 20 h.


VEHICLE CONTROL PERFORMED: yes, comprising fortified groundwater amended with distilled water at a rate of 10 mL/L, corresponding to the maximum stock "solution" addition at the highest 'basisches Chromsulfat' concentration.


RANGE-FINDING STUDY
- Test concentrations: not stated.
- Results used to determine the conditions for the definitive study: yes, but results not stated.
Reference substance (positive control):
no
Remarks:
Not relevant.

Results and discussion

Effect concentrationsopen allclose all
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
: 'basisches Chromsulfat'
Basis for effect:
time to hatch
Remarks:
, hatch rate, survival, growth and behaviour
Remarks on result:
other: None.
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
0.018 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Remarks:
chromium
Basis for effect:
time to hatch
Remarks:
, hatch rate, survival, growth and behaviour
Remarks on result:
other: Highest mean of dissolved Cr concentrations (measured in fresh and expired media at the lowest concentration of 'basisches Chromsulfat' tested).
Details on results:
- Mortality/survival at embryo, larval and juvenile stages: see results table below. There were no statistically significant reductions (one-tailed Student's t-test, p=0.95) in any of these parameters in the groups exposed to 'basisches Chromsulfat', relative to the unamended control.
- Days to hatch or time to release of young: All eggs hatched within 4 days in the two control groups and all the 'basisches Chromsulfat' treatments.
- Numbers hatched: see results table below.
- Number of fish in swim-up stage at one or more time periods (e.g., day x1, x2):
- Observations on body length and weight of young: see results table below. Juvenile fish in the distilled water-amended control were significantly heavier (wet and dry weight) than in the unameded control at test termination.
- Number of healthy fish at end of test: see results table below.
- Type of and number with morphological abnormalities: A maximum of eight larvae per treatment had a slightly swollen broodpouch and/or curved spine in the period between day 7 and day 16 and similar observations were also recorded in the control treatments.
- Type of and number with behavioural abnormalities: none.
- Type and number of developmental effects: none.
- Type and magnitude of hormonal changes: not investigated.
- Other biological observations: all surviving larvae swam and fed actively during the exposure period and no treatment-related malformations were noted at the end of the test.
- Effect concentrations exceeding solubility of substance in test medium: yes. Exposure expressed in terms of nominal concentrations of 'basisches Chromsulfat' (all above solubility limit) as well as measured, dissolved Cr.
- Incidents in the course of the test which might have influenced the results: none.
Results with reference substance (positive control):
Not relevant.
Reported statistics and error estimates:
Not relevant.

Any other information on results incl. tables

Results of analyses of dissolved chromium in fresh and expired media samples are presented below:

Nominal ‘basisches Chromsulfat’ concentration, mg/L

Nominal Cr concentration, mg/L

Measured concentrations of dissolved Cr in filtered samples (mg/L)

d 2

fresh

d 4 expired

d 14

fresh

d 16 expired

d 28

fresh

d 30 expired

mean

0 (gw)

0

<0.002

0.008

0.003

<0.002

<0.002

<0.002

-

0 (gw + dw)

0

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

-

3.2

0.56

0.018

0.004

0.025

0.008

0.036

0.014

0.018

10

1.76

0.016

0.003

0.025

0.007

0.042

0.015

0.018

32

5.6

0.014

0.003

0.022

0.005

0.039

0.014

0.016

100

17.6

0.012

0.003

0.020

0.004

0.024

0.007

0.012

320

56

0.007

0.004

0.005

0.003

0.019

0.004

0.007

1000

176

0.007

0.005

0.006

0.002

0.005

0.004

0.005

gw:         groundwater-based dilution water control.

gw+dw:  groundwater-based dilution water amended with 15 mL distilled water per 1.5 L 

n.s.: not sampled.

Data on hatching, mortality and juvenile growth of B. rerio exposed to 'basisches Chromsulfat' are presented below:

Nominal ‘basisches Chromsulfat’ concentration, mg/L

Eggs hatched after 4 days (%)

Mortality after 30 days
(%)

Number of surviving fish

length

(mm)

wet weight (mg)

dry weight (mg)

0 (gw)

100

6

66

1.3

14.6

3.3

0 (gw + dw)

99

10

64

1.4

18.0*

4.1*

3.2

100

11

62

1.4

17.8

3.8

10

100

12

61

1.4

20.0

4.5

32

100

6

66

1.3

15.7

3.5

100

100

9

64

1.3

14.4

3.1

320

100

7

65

1.3

16.3

3.4

1000

100

10

63

1.3

16.6

3.6

gw:  groundwater-based dilution water control.

gw+dw:  groundwater-based dilution water amended with 15 mL distilled water per 1.5 L 

*  Significantly greater than in the unamended dilution water control. 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The long-term toxicity of 'basisches Chromsulfat' (chromium hydroxide sulphate) to the zebra fish (B. rerio), was determined in a 30-day early life-stage study conducted under semi-static conditions with 48-72 h renewal of pH-adjusted media. The NOEC was 1000 mg/L (nominal), based on the absence of adverse treatment-related effects on hatch, survival, growth and behaviour of juvenile fish at all the concentrations of 'basisches Chromsulfat' applied in the test.

The solubility of 'basisches Chromsulfat' was low under the conditions of the test. Based on the highest mean measured concentration of dissolved chromium at any of the concentrations of 'basisches Chromsulfat' applied in the test, the 30-day NOEC was 0.018 mg Cr/L.
Executive summary:

A study of the long-term toxicity of 'basisches Chromsulfat' (chromium hydroxide sulphate) to fish was conducted in a 30-day early life-stage test with embryonic and hatched juvenile zebra fish (B. danio) exposed to nominal concentrations of 'basisches Chromsulfat' ranging from 3.2 to 1000 mg/L under semi-static conditions, with renewal of pH-adjusted media at 48 -72-h intervals. The 30 -day NOEC of 'basisches Chromsulfat' was 1000 mg/L, based on the highest nominal concentration applied in the test and the absence of adverse effects on hatch, survival, growth and behaviour of the test organisms. The solubility of 'basisches Chromsulfat' was low under the conditions of the test. Based on the maximum mean measured concentration of dissolved chromium recorded in any of the 'basisches Chromsulfat' treatments, the 30 day NOEC was 0.018 mg Cr/L.

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