Hydroxylammonium chloride

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 - 06 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

GLP compliance:

yes (incl. QA statement)

Remarks:

Department of health of the government of the United Kingdom

Analytical monitoring:

yes

Details on sampling:

- Concentrations and sampling method: 10 mL samples were taken from the control and each surviving test concentration at 0 (fresh media), 24, 48, 72 (old and fresh media) and 96 h (old media) for analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 250 mg of the test substance was dissolved in a final volume of 1000 mL of dilution water to give a 250 mg/L stock solution. The pH was adjusted to pH ≥ 6 using 2M NaOH. Aliquots (22.4, 40, 72, 128, 224 and 400 mL) of the stock solution were each separately diluted in a final volume of 10 L of dilution water to give the final concentrations.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: rainbow trout
- Method of holding: fish were held in a temperature controlled room under artificial light, with a 16-h light: 8-h dark photo-period, in holding tanks under continuous water renewal (flow-through) conditions. The water used for the holding of fish stocks and for the toxicity tests was laboratory mains supply. The water was pumped to the laboratory through an activated carbon filter.

ACCLIMATION
- Acclimation period: at least 12 d prior to testing
- Type and amount of food during acclimation: fish were fed with a proprietary fish food, which was added to the holding tank in quantities dictated by the size of the fish. Uneaten food and debris was siphoned or cleaned from the tanks as required.
- Feeding frequency during acclimation: fish were not fed for at least 24 hours before the start of the definitive test
- Health during acclimation (any mortality observed): no mortalities

FEEDING DURING TEST
- No feeding during test

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

14.1 - 15.2 (new media)
14.7 - 16.2 (old media)

pH:

7.04 - 7.35 (new media)
7.08 - 7.78 (old media)

Dissolved oxygen:

91 - 104 % ASV (9.34 - 11.15 mg/L) (new media)
76 - 102 % ASV (7.71 - 10.76 mg/L) (old media)

Nominal and measured concentrations:

Nominal: 0.56, 1.0, 1.8, 3.2, 5.6 and 10 mg/L
Geom. mean meas.: 0.484, 0.834, 1.50, 2.48, 5.03 and 10.3 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 20 L glass aquaria
- Type (delete if not applicable): aquaria covered with a clear plastic lid
- Fill volume: 10 L
- Aeration: yes
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.65 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dilution water passed through activated carbon filters
- Culture medium different from test medium: no
- Intervals of water quality measurement: at the start of the test and at approx. 24 h intervals during the test, the media pH, concentration of dissolved oxygen (% air saturation value and mg/L) and the individual test media temperatures were recorded in the freshly prepared and old test media.

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Photoperiod: 16 hour light: 8 hours dark

EFFECT PARAMETERS MEASURED: all fish were observed at ca 3 h and then at 24-h intervals throughout the test. The number of dead fish and those fish exhibiting toxic symptoms or modified behaviour were recorded.

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L (under static conditions)
- Results used to determine the conditions for the definitive study: the study indicated that the LC50 would be between 1.0 and 10 mg/L and that the test substance could be unstable in the test media over a 96 h period

Reference substance (positive control):

not specified

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1.78 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CI: 1.55 – 2.06 mg/L

Details on results:

- Behavioural abnormalities: see any other information on results incl. tables
- Observations on body length and weight: Mean total length: 4.7 cm (4.2-5.4 cm); Mean wet weight: 0.92 g (0.64 – 1.40 g)
- Mortality of control: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: test solutions were colourless throughout the test

Reported statistics and error estimates:

Statistical analysis was performed using the CETIS program v 1.8.6.8. The LC50 values and corresponding 95% confidence limits at 24, 48, 72 and 96 hours were calculated using the Spearman-Kärber method. The LC10 and LC20 values were calculated using linear interpolation.

Sublethal observations / clinical signs:

VALIDITY CRITERIA

Table 1: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality in the control(s) should not exceed 10% (or one fish if less than ten are used) at the end of the test	0% mortality of control	yes
The dissolved oxygen concentration should be >60%	≥76%	yes

ANALYTICAL RESULTS

Analysis of the freshly prepared media resulted in measured concentrations of 70% to 105% of nominal value. Analysis of the corresponding old media resulted in measured concentrations of 72 to 101% of nominal value with the exception of the 1.0 mg/L test concentration at 96 hours which resulted in a measured concentration of 58% of nominal.

Table 2: Analytical results

Nominal concentration (mg/L)	Measured concentration (mg/L)
	0 hours (fresh media)	24 hours (old media)	24 hours (fresh media)*	48 hours (old media)	48 hours (fresh media)*	72 hours (old media)	72 hours (fresh media)*	96 hours (old media)	Geometric mean measured concentration (mg/L)	% nominal value
Control	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
0.56	0.535 (96)	0.429 (77)	0.532 (95)	0.455 (81)	0.492 (88)	0.423 (76)	0.566 (101)	0.461 (82)	0.484	86
1	0.981 (98)	0.852 (85)	0.852 (85)	0.863 (86)	0.879 (88)	0.744 (75)*	1.00 (100)	0.581 (58)**	0.834	83
1.8	1.65 (92)	1.65 (92)	1.54 (86)	1.44 (80)	1.44 (80)	1.29 (72)*	1.58 (88)	1.41 (78)	1.5	83
3.2	2.67 (83)	2.61 (82)	2.24 (70)	2.43 (76)	NA	NA	NA	NA	2.48	78
5.6	4.91 (88)	5.15 (92)	NA	NA	NA	NA	NA	NA	5.03	90
10	10.5 (105)	10.1 (101)	NA	NA	NA	NA	NA	NA	10.3	103

Values in brackets = % nominal value

* Initial analysis of samples showed unexpected results and were therefore re-analysed. Re-analysis showed measured concentrations comparable to previous old media analyses therefore the initial results were considered to be erroneous and have not been reported

** Samples re-analysed to confirm results. Re-analysis results were comparable to the above results and have therefore not been reported

BIOLOGICAL RESULTS

Further reported effect concentrations:

LC10 (96 h) = 1.28 mg/L

LC20 (96 h) = 1.56 mg/L

NOEC (96 h) = 0.834 mg/L

Table 3: Mortality of fish

	Cumulative number of mortalities recorded (7 fish initially)
Geometric mean measured concentration (mg/L)	24 h	48 h	72 h	96 h
	0	0	0	0
0.484	0	0	0	0
0.834	0	0	0	0
1.5	0	1	1	1
2.48	5	7	7	7
5.03	7	7	7	7
10.3	7	7	7	7

The temperature did vary by more than 1ºC in some instances. This deviation was considered not to affect the integrity of the study given that no adverse effects were observed in the control groups and the temperature remained within the 15 ± 2ºC range throughout the tests.

Table 4: Sub-lethal effects recorded during the test

Observation time	Geometric mean measured concentration (mg/L)	Observation
4 h	10.3	Mild hyperventilation
6 h	10.3	3 fish – barrel rolling and overturned at base of vessel
		4 fish – orientation issues
7 h	10.3	4 fish – overturned at base of vessel
22 h	5.03	All dead
	2.48	2 fish – dead
		1 fish - barrel rolling and overturned at base of vessel
24 h	2.48	2 fish – overturned at surface
28 h	2.48	1 fish – dead
		1 fish – hyperventilating/head shaking/orientation issues
30 h	2.48	1 fish - barrel rolling and overturned at surface
48 h	1.5	1 fish barrel rolling

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

LC50 (96 h) = 1.78 mg/L (geom. mean meas., Oncorhynchus mykiss, OECD 203)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

1.78 mg/L

Additional information

One study is available for the substance hydroxylammonium chloride (CAS 5470-11-1), in which the short term toxicity to fish is tested. The study was conducted according to OECD guideline 203 and GLP. The test organism, Oncorhynchus mykiss was exposed in a semi-static freshwater system to nominal test item concentrations of 0.56, 1.0, 1.8, 3.2, 5.6 and 10 mg/L (geom. mean meas.: 0.484, 0.834, 1.50, 2.48, 5.03 and 10.3 mg/L) for 96 h. The test media was renewed daily during the test. The test concentrations were monitored at time points 0 (fresh media), 24, 48, 72 (old and fresh media) and 96 h (old media) by LC-TOF/MS. The measured concentrations ranged between 58 and 105% of the nominal values. Therefore the effect values were expressed as geom. mean meas. concentrations. The 96 h LC50 was determined at 1.78 mg/L (geom. mean meas.).