Reaction mass of chromium (+ 3) hydroxide sulfate and sodium sulfate

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988-07-28 - 1988-08-12 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

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.

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

Analytical monitoring:

yes

Details on sampling:

- Concentrations: nominal 3.2, 10, 32, 100, 320 and 1000 mg/L
- Sampling method: 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 about 0.5 hours after adjusting the pH to that of the dilution water. 100 mL samples were taken from one of each pair of duplicate test vessels of the d 2, d 14 and d 28 (fresh) and d 4, d 16 and d 30 (expired) media of the control and each treatment group.
In addition about 5 mL samples were taken of the concentrated stock solutions containing 100 g test material per L of distilled water, which were prepared five times throughout the test duration and diluted prior to analysis to detemine total Cr.
- Sample storage conditions before analysis: The samples taken at t = 2 d, t = 14 d, t = 16 d, t = 28 d and t = 30 d were filtered (0.45 µm membrane filter) immediately and then acidified to determine concentrations of dissolved Cr, with the exception of d 4 expired media samples which were not processed until 3 days after collection.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A concentrated stock solution containing 100 g test materail/L of distilled water was prepared every week by weighing accurately 20.0 gram of test material and dissolving this in 200 mL of distilled water. This concentrated stock solution was clear and green. From the concentrated stock solution 0.048, 0.15, 0.48, 1.5, 4.8 and 15 mL were diluted to 1500 mL with dilution water and the pH was adjusted to the pH of the dilution water. These solutions containing nominal 3.2,10, 32,100, 320 and 1000 mg/L were used as test solutions.
- Controls: dilution water (synthetic water, for detailed composition please refer to "any other information on materails and methods incl. tables"); because distilled water was used to dose the test substance stock solutions, an extra control solution was prepared containing 1.5 mL of distilled water per 1.5 L, this being the highest amount of distilled water used for dosing.
- Test concentration separation factor: 3.2
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Before adjusting the pH the test solutions of 3.2, 10, 32, 100 and 320 mg/L were turbid with a greenish colour, the intensity of the green colour increased with increasing concentration. The test solution of 1000 mg/L was a clear greenish solution. After adjusting the pH and after standing for about 0.5 hour the test solutions of 3.2 mg/L and 10 mg/L were turbid (10 mg/L more so than 3.2 mg/L); the test solution of 32 mg/L was even more turbid than that of 10 mg/L and contained undissolved material in the solution and on the bottom; the test solutions of 100 and 320 mg/L also contained undissolved material in the solutions and on the bottom of the test vessels; the test solution of 1000 mg/L contained undissolved material on the bottom of the test vessel, but no undissolved material in the solution. The amount of (blue/green) undissolved material on the bottom of the test vessels of the test solutions containing 32, 100, 320 and 1000 mg/L increased with increasing concentrations.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebrafish
- Species: Brachydanio rerio
- Source: Adult fishes were obtained from the commercial tropical fish hatchery M.B. Ruijsbroek B.V. (Noord-vliet 159, Maassluis), the Netherlands.
- Age at study initiation (mean and range, SD): fertilised eggs; the developmental stage of the eggs at the start of the test was the young blastula stage of fertilised eggs (verified by microscopy)
- Method of breeding: Before the first controlled spawning in the laboratory females and males were separated and placed in basins at 24°C. The females were kept under a 7 h light / 17 h dark regime and were held individually to reveal their spawning history (frequency of spawning, condition of eggs). The males were kept under the environmental daylight and dark.
At 06.00 h about a week after a previous spawning in the laboratory, one female (carrying young eggs) was put into a test basin at 26°C in which three males had been present since 16.00 h the previous day. As soon as the eggs were laid they were collected and put into the test basins. This was within 6 h of spawning.
- Feeding during test
- Food type: As soon as the first fish larvae were seen the fishes were fed abundantly with rotifers (Brachionus rubens), cultured in mass cultures in the laboratory and from t = 10 d, in addition, with Artemia nauplii enriched with Selco.

ACCLIMATION
- Acclimation period: three weeks
- Acclimation conditions (same as test or not): temperature of 24°C, dilution water as culture medium
- Type and amount of food: They were fed with Artemia nauplii (enriched with Selco) and fresh minced steak.
- Feeding frequency during acclimation: daily

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): To start the test 6 batches of eggs (from 6 females) were needed; they were equally divided (per batch) between all test and control solutions.
- 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. This procedure was followed because it is desirable to expose the eggs as soon as possible after fertilization, when it is not yet possible to see if the eggs have indeed been fertilized. The hatching and survival of these batches of about 35 eggs/larvae were followed during the remaining exposure time.

POST-HATCH FEEDING
- Start date: As soon as the first fish larvae were seen.
- 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

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.

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 21samples left unaerated - with food detritus but without fish - for a long time after media renewal.

Salinity:

Not relevant.

Nominal and measured concentrations:

nominal: 3.2, 10, 32, 100, 320 and 1000 mg/L and a control
mean measured test concentrations: 0.018, 0.018, 0.016, 0.012, 0.007 and 0.005 mg Cr/L (the test material was was apparently almost insoluble in the dilution water used and most of the dosed amount immediately precipitated)

Details on test conditions:

TEST SYSTEM
- Test vessel: beakers
- Type (delete if not applicable): covered with a watch 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 test material, 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: At the start of the test at least 80 eggs, probably just fertilized, were placed in each test vessel. After the first 24 h of exposure this number was reduced to yield about 35 fertilized eggs per vessel.
- No. of vessels per concentration (replicates): two
- No. of vessels per control (replicates): two
- No. of vessels per vehicle control (replicates): two

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Groundwater obtained near Linschoten, the Netherlands, fortified with mineral salts, prepared in two 10,000 L batches for this study. For complete information on the composition of the dilution wtarer, please refer to section "Any other information on materails and methods incl.tables".
- Total organic carbon: 1.09-1.14 mg/L.
- Ca/Mg ratio: 1.76-1.88 (mol/mol)
- Culture medium different from test medium: no
- 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
- Light intensity: not stated

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The 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 study termination. Length and wet weight of individual survivors was determined at test termination and group-mean dry weights were determined after drying at 60 °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.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.125

Reference substance (positive control):

no

Remarks:

Not applicable.

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: There were no statistically significant effects (one-tailed Student's t-test, p=0.95) in the treatment groups exposed to the test material, relative to the unamended control. Accordingly, mortality of eggs or larvae of Brachydanio rerio was not affected by the test material at nominal concentrations up tp and including 1000 mg/L during an exposure of 30 days after placing the newly fertilized eggs in the test solutions.
- 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. Accordingly, hatching of eggs or larvae of Brachydanio rerio was not affected by the test material at nominal cponcentrations up to and including 1000 mg/L.
- Numbers hatched: Please refer to the table presented in section "Any other information on results incl. tables".
- Observations on body length and weight of young: Juvenile fish in the distilled water-amended control were significantly heavier (wet and dry weight) than in the unameded control at test termination. However, no significant growth retardation was determined at the nominal test concentrations up to and including 1000 mg/L.
- 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. However, no malformations or morphological abnormalities were determined in the treatment groups up to and including the nominal test concentration of 1000 mg/L.
- 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. Most of the dosed amount immediately precipitated at the bottom of the test beakers.
- 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:

One-tailed Student's t-test, p=0.95

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.

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to a draft version of OECD 210 on the registered substance itself, without deviations which may have an impact on the validity of the study. Thus, the results were obtained via a scientifically reasonable method and the obtained results can be considered as reliable to assess the long-term toxicity of the test substance towards fish.
In a semi-static test fertilised eggs of the zebrafish (Brachydanio rerio) were exposed to the test substance at the concentrations of nominal 3.2, 10, 32, 100, 320 and 1000 mg/L (on basis of total chromium) and a control for a period of 30 days (26 days post-hatch) under defined conditions. Using electrothermal atomisation atomic absorption spectrometry (AAS detection) arithmetic mean measured concentrations of 0.0008, 0.0045 and 0.015 mg Cr/L (dissolved chromium) were determined.
Based on the obtained results no effects were observed on hatch, survival, growth and behaviour of juvenile fish at all the concentrations of test material applied in the test. The 30-day NOEC values for hatch, survival, growth and behaviour were therefore determined to be greater than nominal 1000 mg/L, corresponding to ≥ 0.018 mg total Cr/L, based on mean measured concentrations of dissolved chromium, respectively.

Executive summary:

The long-term toxicity of the registered substance to fish was tested in a 30-day early life-stage test conducted with embryonic and hatched juvenile zebrafish (Brachydanio rerio) to determine the influence on hatching, mortality and presence of malformations of eggs and larvae as laid down in the Draft OECD TG 210, with an exposure period of 30 days (26 days post-hatch) under defined conditions.The test was carried out as a semi-static test, with replacement of the test solutions 2 -3 times per week. The concentrations tested were nominal 3.2, 10, 32, 100, 320 and 1000 mg of test material added per L dilution water. The test substance as supplied contained 80% Cr2(OH)2(SO4)2*Na2SO4.

During the test samples of the supernatant in the test solutions were taken three times just after dosing (freshly prepared) and three times just before the next replacement (spent/aged); these samples were analysed for chromium after filtration and acidification. Using electrothermal atomisation atomic absorption spectrometry (AAS detection) arithmetic mean measured concentrations of 0.0008, 0.0045 and 0.015 mg Cr/L (dissolved chromium) were determined.

Based on the obtained results no effects were observed on hatch, survival, growth and behaviour of juvenile fish at all the concentrations of test material applied in the test. The 30-day NOEC values for hatch, survival, growth and behaviour were therefore determined to be greater than nominal 1000 mg/L, corresponding to ≥ 0.018 mg total Cr/L, based on mean measured concentrations of dissolved chromium, respectively.

Description of key information

Key_Long-term toxicity to fish: NOEC (26d) ≥ 0.018 mg Cr/L (arithmetic mean measured) for Brachydanio rerio (semi-static with media renewals at intervals of 2-3 days, freshwater, Method of the German Umweltbundesamt (UBA), Berlin of May 1984, equivalent to the draft version of OECD TG 210 of 1988, GLP)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Remarks:

based on dissolved chromium

Effect concentration:

>= 0.018 mg/L

Additional information

The long-term toxicity of the registered substance to fish was tested in a 30-day early life-stage test conducted with embryonic and hatched juvenile zebrafish (Brachydanio rerio) to determine the influence on hatching, mortality and presence of malformations of eggs and larvae as laid down in the Draft OECD TG 210, with an exposure period of 30 days (26 days post-hatch) under defined conditions.The test was carried out as a semi-static test, with replacement of the test solutions 2 -3 times per week. The concentrations tested were nominal 3.2, 10, 32, 100, 320 and 1000 mg of test material added per L dilution water. The test substance as supplied contained 80% Cr2(OH)2(SO4)2*Na2SO4.

During the test samples of the supernatant in the test solutions were taken three times just after dosing (freshly prepared) and three times just before the next replacement (spent/aged); these samples were analysed for chromium after filtration and acidification.Using electrothermal atomisation atomic absorption spectrometry (AAS detection) arithmetic mean measured concentrations of 0.0008, 0.0045 and 0.015 mg Cr/L (dissolved chromium) were determined.

Based on the obtained results no effects were observed on hatch, survival, growth and behaviour of juvenile fish at all the concentrations of test material applied in the test. The 30-day NOEC values for hatch, survival, growth and behaviour were therefore determined to be greater than nominal 1000 mg/L, corresponding to ≥ 0.018 mg total Cr/L, based on mean measured concentrations of dissolved chromium, respectively.