Ascophyllum nodosum, ext.

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August-September 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: in accordance with guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ascophyllum nodosum, ext.

EC Number:

283-907-6

EC Name:

Ascophyllum nodosum, ext.

Cas Number:

84775-78-0

Molecular formula:

Not applicable; a generic molecular formula cannot be provided for this UVCB substance.

IUPAC Name:

Ascophillum nodosum, extract

Details on test material:

- Name of test material (as cited in study report): Algea Fert Solid K+
- Substance type: fertilizer ( algal extract)
- Physical state: solid, black micro-flakes
- Lot/batch No.: 00680
- Expiration date of the lot/batch: March 2012
- Stability under test conditions: Stable under suitable storage conditions.
- Storage condition of test material: original tightly-closed containers in well- ventilated place.

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

The test medium was prepared as follows: prior to the start of the test, 1 litre of a test item stock solution of 2 g/L was prepared by direct weighting into reconstituted water (2.0001 g of test item into 1000 mL of reconstituted water). The obtained solution appeared dark-brown; it was manually mixed and then it was mixed by the mean of a magnetic stirrer for about 1 hour to achieve the totally solubilisation of test item.
The solution was then poured into the test tank to achieve the volume of 20 L.
The test solution and the control were let to reach the desired temperature before adding the fish.

Test organisms

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebra fish
- Source: Il Grande Blu - Milan
- Age at study initiation : juvenile fish
- Length at study initiation : 1.8 cm ( mean value)
- Weight at study initiation (mean and range, SD): 0.25 g (mean value)
- Feeding during test: none
- Food type: commercial fish food (Tetramin granules) until 48 hours before the test initiation.


ACCLIMATION
- Acclimation period: at least 12 days at the same conditions regarding water quality, temperature and illumination, as during the test
- Acclimation conditions : same as for test
- Health during acclimation (any mortality observed): there was no mortality during the acclimation period.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Test conditions

Hardness:

Water Hardness: 10 - 250 mg/L as CaCO3

Test temperature:

The water temperature in aquaria was measured twice by the mean of a digital thermometer . The test temperature was between 22.3 and 22.9 °C for control tank and between 22.3 and 23.0 °C for the test item solution tank.
Photoperiod: the test was performed under a 16 hours light and 8 hours darkness daily photoperiod; light intensity was 50 - 500 Lux, with a 30-minutes transition period (OPPTS 850.1075). Light intensity was daily measured and it was between 118 and 132 Lux.

pH:

The pH values in test item solution and in the control were in the range 7.05 – 7.39, as required by the above mentioned guidelines and did not vary more than 0.20 units between the start and the end of test period.
The pH of the test water after equilibration was in the range 7.11 – 7.29 and did not vary more than 1.5 units during the single test.

Dissolved oxygen:

During the test period, the dissolved oxygen concentrations in the test medium and the control were always above 60 per cent of the air saturation value

Salinity:

CaCl2 x 2H2O :2.0 mmol/L (= 294 mg/L)
MgSO4 x 7H2O:0.5 mmol/L (= 123 mg/L)
NaHCO3: 0.75 mmol/L (= 65 mg/L)
KCl:0.075 mmol/L (= 5.8 mg/L)
Alkalinity: 0.8 mmol/L
Ratio of Ca : Mg = 4 : 1 (based on molarity), Na : K= 10 : 1 (based on molarity)

Nominal and measured concentrations:

100 mg/L (nominal).

Details on test conditions:

The test concentration was chosen based on the results of a pre-test (range-finding test), which was not performed in compliance with the GLP Regulations. The study was carried out at the only nominal product concentration of 100 mg/L. Moreover, one tank with no test item was also prepared as the negative control.
For the test concentration and for the negative control, 60 L capacity glass aquaria were used. The loading rate did not exceed 0.8 g (fresh weight) of fish per litre of test solution (OPPTS 850.1075) and one replicate per concentration was used. At the start of the test, 7 fish were randomly distributed to each aquarium. The test was performed in single temperature-controlled aquarium and incubated in a temperature-controlled room.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 2

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 3

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

The fish were checked for mortality and abnormalities in appearance and behaviour after 2, 24, 48, 72 and 96 hours.
In the control 0% mortality was observed during the test period. This value complies with the validity criterion reported in the guidelines (maximum mortality of one fish, if seven fish were used, in the control medium at the end of the test).
The NOEC and the LOEC were directly determined from the Raw Data by statistical comparison with the control (Fisher’s Exact test) at each observation time.

The NOEC (highest concentration tested without toxic effects after the exposure period) of Algea Fert Solid K+ to zebra fish was determined to be equal to the tested concentration, 100.0 mg/L.
Based on the observed effects, the LOEC and the 96-h LC50 were determined to be higher than 100.0 mg/L.

Details of the statistical analysis are reported in Appendix B.

During the test period any behaviour and/or pathological signs on fishes exposed to the test item and in the control were observed.

As for the test medium appearance, no remarkable observations were made. The test medium was a dark-brown solution throughout the test duration.

Reported statistics and error estimates:

The NOEC and the LOEC were determined from the Raw Data by statistical comparison with the control (Fisher’s Exact test) at each time observations.
The CETIS v.1.026D software was used to carry out the statistical analyses, using nominal concentration as input data.

Any other information on results incl. tables

Sublethal observations / clinical signs:

Lethal effect of Algea Fert Solid K+ to zebra fish

Nominal test item concentration [mg/L]	n. of exposed fish	n. dead fish
		2 h	24 h	48 h	72 h	96 h
Negative control	7	0	0	0	0	0
100.0	7	0	0	0	0	0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

A test is not acceptable if more than one control fish dies during the 96-hour test period. Dissolved oxygen concentration shall be maintained above 60 per cent of the air saturation value.

Conclusions:

The test item Algea Fert Solid K+ caused 0% mortality to zebra fish after 96-hours exposure at the nominal product concentration of 100 mg/L in a limit static test.
Given the nature of limit test, the 96-hour LC50 could not be determined, but, according to the binomial theory, using 7 fishes per test the absence of mortality provides at least 99% confidence that the LC50 is greater than the tested concentration.

After 96 hours of exposure, the NOEC, LOEC and LC50 values, calculated based on the nominal concentration, were assessed to be as follows:

96 h NOEC: 100.0 mg/L
96 h LOEC: > 100.0 mg/L
96 h LC50: > 100.0 mg/L

Executive summary:

 The acute toxicity of the test item Algea Fert Solid K+ to zebra fish (Danio rerio)was determined in a limit, 96‑hour static test according to the following guideline:

- OECD Guideline for Testing of Chemicals, N. 203. “Fish, acute toxicity test”, 1992,

- OPPTS 850.1075 guideline “Fish acute toxicity test, freshwater and marine”, EPA Ecological effects Test guidelines, 1996.

 For this purpose, juvenile fish of the speciesDanio reriowere exposed, after an acclimation period, to an aqueous test medium containing the test item at the only nominal concentration of 100.0 mg/L. Besides this test concentration, one tank without the test item was prepared as the negative control.

The test organisms were checked for mortality after 2, 24, 48, 72 and 96 hours from the test initiation.

 The pH values of the test medium after preparation was 7.29 for test item and 7.11 for control. Therefore, the pH of the test medium was not adjusted before of the test starting.

 

Throughout the test, the dissolved oxygen was all the time above 60 per cent of the air saturation value both in the control and in the test solution, as provided by OECD guideline.

 

The test temperature was between 22.3 andfor control tank and between 22.3 andfor the test item solution tank, according to the OECD’s recommended range (23.0±2.0°C).

 

Light intensity during the test period was daily measured and it was in the range 118 – 126 for control tank and between 126 and 132 Lux for test item solution tank, within the range 50-500 Lux.

 In the control no mortality was observed during the test period. This value complies with the validity criterion reported in the guidelines (maximum mortality of one fish, if seven fish were used, in the control medium at the end of the test).

 No mortality was observed in the test item medium after 96 hours, therefore the NOEC (highest concentration tested without toxic effects after the exposure period of 96 hours) of Algea Fert Solid K+ to zebra fish was determined to be equal to the tested nominal concentration of 100 mg/L (Fisher’s exact test).

Based on the observed effects, the LOEC and the 96-h LC₅₀were determined to be greater than the nominal tested concentration, 100.0 mg/L.

 During the test period, any behaviour and/or pathological signs on fishes exposed to the test item and in the control were observed.