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Working for a better future

REPORT

: Acute Toxicity to Rainbow Trout

HLS study number: CEF0010

Version ID: Audited Draft 1 Report

Issue date: 10 October 2013
Huntingdon Life Sciences: CEF0010

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Sponsor and Test Facility Details

Sponsor

Test facility Huntingdon Life Sciences

Eye Research Centre

Eye

Suffolk

IP23 7PX

UK
Huntingdon Life Sciences: CEF0010

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Table of Contents

Sponsor and Test Facility Details.......................................................................................... 2

Compliance with Good Laboratory Practice .......................................................................... 5

Quality Assurance Statement................................................................................................ 6

Contributing Scientists .......................................................................................................... 7

Summary .............................................................................................................................. 8

1. Introduction ................................................................................................................... 9

2. Test Substance ........................................................................................................... 10

3. Experimental Procedure.............................................................................................. 11

3.1 Test species.......................................................................................................... 11

3.1.1 Name............................................................................................................. 11

3.1.2 Source ........................................................................................................... 11

3.1.3 Acclimatisation............................................................................................... 11

3.2 Diluent water......................................................................................................... 11

3.3 Test substance preparation................................................................................... 12

3.4 Exposure conditions.............................................................................................. 12

3.4.1 Experimental design ...................................................................................... 12

3.4.2 Test concentrations........................................................................................ 12

3.4.3 Medium renewal ............................................................................................ 12

3.4.4 Stability of test concentrations ....................................................................... 12

3.4.5 Environmental conditions............................................................................... 13

3.5 Criteria of effect..................................................................................................... 13

3.6 Evaluation of data ................................................................................................. 13

3.7 Protocol deviations................................................................................................ 13

4. Maintenance of Records.............................................................................................. 14

5. Results........................................................................................................................ 15

5.1 Chemical analysis................................................................................................. 15

5.2 Mortality and observations .................................................................................... 15

5.3 Environmental parameters .................................................................................... 15

6. Conclusions................................................................................................................. 16

List of Tables

Table 1 Measured concentrations.............................................................................17

Table 2 Sub-lethal effects .........................................................................................18

Table 3 Environmental parameters ...........................................................................19
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List of Appendices

Appendix 1 Certificate of analysis...................................................................................20

Appendix 2 Typical water quality characteristics of the diluent supply.............................21

Appendix 3 The determination of in dechlorinated tap water..........................22

Appendix 4 Compliance statement .................................................................................32
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Compliance with Good Laboratory Practice

: Acute Toxicity to Rainbow Trout

The study described in this report was conducted in compliance with the following Good 

Laboratory Practice standards and I consider the data generated to be valid.

The UK Good Laboratory Practice Regulations (Statutory Instrument 1999 No. 

3106, as amended by Statutory Instrument 2004 No. 994).

OECD Principles of Good Laboratory Practice (as revised in 1997), 

ENV/MC/CHEM (98) 17.

EC Commission Directive 2004/10/EC of 11 February 2004 (Official Journal No L 

50/44).

These principles of Good Laboratory Practice are accepted by the Regulatory Authorities of

the United States of America and Japan on the basis of Intergovernmental Agreements.

No claim of compliance is made for the results of analysis of the diluent water supply.

___________________________________________ ________________________

Romalie Allen BSc Date

Study Director

Huntingdon Life Sciences
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Quality Assurance Statement

: Acute Toxicity to Rainbow Trout

The following inspections and audits have been carried out in relation to this study:

Study Phase Date(s) of Inspection Date of Reporting to Study 

Director and Management

Protocol Audit 13 Jun 2013 13 Jun 2013

Report Audit 10 Oct 2013 10 Oct 2013

Process based inspections: At or about the time this study was in progress inspections of

procedures employed on this type of study were carried out. These were conducted and 

reported to appropriate Company Management as indicated below:

Process Based Inspections Date(s) of Inspection Date of Reporting to 

Management

Environmental measurements & 

monitoring/Ammonia analysis 

07 Mar 2013 07 Mar 2013

Study management & conduct 26 Jun 2013 26 Jun 2013

Sampling of test media for chemical 

analysis

09 Jul 2013 09 Jul 2013

Dose Formulation 09 Jul 2013 09 Jul 2013

Study management & conduct 09 Jul 2013 09 Jul 2013

Experimental Set-up 11 Jul 2013 11 Jul 2013

Experimental Set-up 08 Aug 2013 09 Aug 2013

Sampling of test media for chemical 

analysis 

08 Aug 2013 09 Aug 2013

Dose Formulation 08 Aug 2013 16 Aug 2013

Study management & conduct 09 Aug 2013 16 Aug 2013

Observations 13 Sep 2013 13 Sep 2013

Measurements 10 Oct 2013 10 Oct 2013

Standard preparation & Fortification 

procedures 

05 Aug 2013-

06 Aug 2013

07 Aug 2013

Results Processing 30 Aug 2013 30 Aug 2013

Chromatography 30 Aug 2013 30 Aug 2013

Sample preparation (to include 

sample management & tracking) 

30 Sep 2013 30 Sep 2013

In addition, an inspection of the facility where this study was conducted was carried out on an 

annual basis. These inspections were reported to Company Management.

........................................................................................ .....................

T Pullinger MRQA Date

Group Manager

Department of Quality Assurance

Huntingdon Life Sciences 
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Contributing Scientists

: Acute Toxicity to Rainbow Trout

Study management 

Aquatic Ecotoxicology and Biodegradation

Romalie Allen BSc

Study Director

Expert department

Environmental Analysis

Simon Tate Higher National Certificate (Applied Chemistry)

Senior Study Manager
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Summary

A study was performed to assess the acute toxicity of to rainbow trout 

(Oncorhynchus mykiss) under semi-static conditions with daily renewal of the media. 

The study was conducted in accordance with EC Methods for Determination of Ecotoxicity, 

Annex to Commission Regulation (EC) No 440/2008 (O.J. No. L142/446, 2008) Part C, 

Method 1 “Acute Toxicity For Fish” and the OECD Guideline for Testing of Chemicals No. 

203 “Fish, Acute Toxicity Test” (1992).

Groups of seven juvenile fish were exposed to , dispersed in water at nominal 

concentrations of 1.17, 2.58, 5.68, 12.5 and 27.5 mg/L. The test media were prepared by

direct addition of the test substance to water for the 27.5 mg/L preparation and by dilution of 

this high concentration (aliquots of 850 mL, 1880 mL, 4.13 L and 9.09L) as appropriate. 

At all concentrations, the intended exposure levels of were achieved between 70 

to 98% of their nominal values. The slightly low concentrations are considered to have 

resulted from a slightly lower solubility at the lower temperature for an acute fish study when 

compared with the physiochemical properties study (20oC with approx. 15oC for this fish 

study). The concentrations were maintained during the test (between 79 to 136% of their 

starting values). Based on a geometric mean, overall mean measured levels were 0.950, 2.19, 

4.65, 10.5 and 24.5 mg/L and these values have been used in the determination of study endpoints.

Observations of the fish were made after approximately 2, 4, 24, 48, 72 and 96 hours of 

exposure.

No mortality occurred during the 96 hour test, therefore the 96 hour LC50 was greater than 

24.5 mg/L under the conditions of this study.

The ‘no-observed effect concentration’ (NOEC) was 10.6 mg/L as treatment-related effects 

(loss of body colour, loss of co-ordination and/or spiral swimming) were exhibited at 

measured concentrations 24.5 mg/L at 24 hours.
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1. Introduction

This study was designed to assess the acute toxicity of to rainbow trout 

(Oncorhynchus mykiss) under semi-static conditions.

The study was conducted in accordance with EC Methods for Determination of Ecotoxicity, 

Annex to Commission Regulation (EC) No 440/2008 (O.J. No. L142/446, 2008) Part C, 

Method 1 “Acute Toxicity For Fish” and the OECD Guideline for Testing of Chemicals No. 

203 “Fish, Acute Toxicity Test” (1992).

The protocol was approved by Huntingdon Life Sciences Management and the Study

Director on 17 June 2013 and by the Sponsor on 1 October 2013.

The study was conducted at Huntingdon Life Sciences, Eye Research Centre, Eye, Suffolk, 

IP23 7PX, England.

The experimental start and completion dates of the study were 25 June 2013 and

30 August 2013, respectively.

A study to determine the physiochemical properties of (HLS study: CEF0001) 

indicated that the limit of solubility of the test substance was 27.5 mg/L at 20oC.
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2. Test Substance

Name

Chemical name

CAS No.

Physical state Powder

Batch number 12342

Purity To be confirmed

Storage Room temperature in the dark

Expiry Date To be confirmed

Date received: 4 March 2013

Certificate of analysis: Appendix 1
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3. Experimental Procedure

3.1 Test species

3.1.1 Name

Rainbow trout (Oncorhynchus mykiss).

3.1.2 Source

The fish were supplied by a commercial fish farm in the UK and were reared at the farm from 

eggs that hatched in March 2013.

3.1.3 Acclimatisation

The stock of fish was obtained from the supplier on 4 July 2013 and they were held in an 

aerated supply of diluent water under flow-through conditions until use. During the 14-day

period immediately before the definitive test, temperatures remained within the range 14.2 to 

14.5C, pH values within the range 7.51 to 7.60, dissolved oxygen concentrations within the 

range 65 to 95% air saturation value (ASV) and total hardness within the range 143 to 

227 mg/L as CaCO3 (see deviations from Protocol section). 

The fish were fed daily with an amount of commercial fish food (TROUW (UK) Ltd; Nutra 

Fry 02) equivalent to between 1 and 4% of the total wet-weight of fish in the holding tank. 

No food was given during the 24-hour period immediately before exposure or during the 

exposure period itself. No medication was given during the holding period, and mortality 

was recorded as <0.5% in the 14 days before the definitive test.

The size of the fish used in the definitive test was determined by weighing and measuring a 

sample of ten fish taken at random from the holding tank on 15 August 2013; their mean total 

length was 6.5 cm and their mean wet weight was 2.52 g.

3.2 Diluent water

The water used to hold the fish and for the study was laboratory tap water, dechlorinated and 

softened by passage through a water purification system. It was passed through a high grade 

activated carbon filter to remove chlorine and any organic contaminants. A proportion of the 

supply then passed through a water softener before final reverse osmosis treatment to produce 

a highly purified water supply. The two grades of dechlorinated water were then remixed to 

give a supply with the desired water hardness. This water was then held in an intermediate 

tank where it was equilibrated to the test temperature and gently aerated before being 

supplied to the holding and test areas. Typical water quality characteristics of the diluent 

supply are given in Appendix 2.
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3.3 Test substance preparation

The method of preparation used during the definitive test was based on the results of a 

formulation trial and a range finding test.

The test substance (1.1 g) was ground into a smooth pourable paste on an agate mortar with 

the dropwise addition of dechlorinated tap water. The paste was then added to a 40 L tank 

containing dechlorinated tap water, made up to volume and stirred for a minimum of one 

hour. The formulation was then filtered (0.45 μm pore size) to provide an aqueous solution 

with a nominal concentration of 27.5 mg/L that was either used directly for the highest test 

concentration (20L) or aliquots (850 mL, 1880 mL, 4.13 L or 9.09L) were diluted in tanks 

(20L capacity) using dechlorinated tap water to provide test media with nominal 

concentrations of 12.5, 5.68, 2.58 and 1.17 mg/L. 

3.4 Exposure conditions

3.4.1 Experimental design 

A range finding test was followed by a definitive test with five test concentrations, plus a 

diluent water control group.

In the definitive test, seven fish were placed at random into each glass aquarium containing 

the prepared control or test media. Each vessel contained 20 litres of medium to a depth of 

19 cm. This provided an initial static loading of 0.882 g bodyweight/Litre.

3.4.2 Test concentrations

The range finding test employed nominal test concentrations of 0.27, 2.7 and 27 mg/L. After 

96 hours, one fish died at 0.27 mg/L, which was considered not to be treatment related since

no mortalities were observed at the two higher test concentrations. At 27 mg/L, there were 

no treatment-related effects noted by 72 hours, but at 96 hours, all fish showed lethargy. No 

significant treatment-related effects were noted in the other test groups. Based on these 

results, the definitive test employed the following nominal concentrations of 1.17, 2.58, 5.68, 

12.5 and 27.5 mg/L; the highest concentration employed approximated to the limit of 

aqueous solubility of the test substance.

3.4.3 Medium renewal

The fish were exposed to the control or test conditions for a period of 96 hours with daily 

batch renewal of the media to ensure the maintenance of satisfactory environmental

conditions and to maintain stable exposure levels.

3.4.4 Stability of test concentrations

The test concentrations of were measured using liquid chromatography with

tandem mass spectrometric detection (LC-MS/MS, see Appendix 3). Two, mid-vessel 

samples (10 mL) of media were taken from the control and test vessels at 0 and 72 hours 

(fresh media) and at 24 and 96 hours (expired media) for analysis. 

On each occasion, one of the samples was analysed and the other was stored in a freezer in 

case further analysis was required.
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3.4.5 Environmental conditions

Treatment and control groups were maintained at 12 to 17C throughout the exposure period 

and constant to within  1C during the study. The temperature of the water in the diluent 

water control vessel was continuously monitored during the study.

Supplementary aeration was provided via narrow bore glass tubes. A photoperiod of 16 

hours light: 8 hours dark was maintained, with periods of subdued lighting at the beginning 

and end of each light phase. Daily records of temperature, pH and dissolved oxygen were 

kept for each control and test vessel together with measurements of total hardness for selected 

vessels at 0 hours. The fish were not fed during the 96 hour exposure period.

3.5 Criteria of effect

The criteria of death employed in this study were (i) absence of respiratory movement and (ii) 

absence of response to physical stimulation of the caudal peduncle.

In addition to observations on mortality at approximately 2, 4, 24, 48, 72 and 96 hours, 

subjective assessments were also made on the incidence and type of any sub-lethal effects 

compared with control fish.

3.6 Evaluation of data

The “no observed effect concentration” (NOEC) was derived by direct inspection of the data 

for lethal and treatment-related-effects. An incidence rate of more than one affected fish out 

of seven is considered to be significant.

3.7 Protocol deviations

The following deviation from Protocol occurred: 

The final study Protocol states that “Fish to be used in the test will be held for at least 

7 days in water of the quality and temperature to be used in the test”. Hardness 

measurements should be approximately 120 to 180 mg/L as CaCO3. On Day -6 the 

hardness measurement was 227 mg/L as CaCO3 for the batch of fish available for use 

on this study. All other recordings for this parameter were within specifications and 

on Day 0 was 164 mg/L as CaCO3. Therefore the fish were considered suitable for 

use on this study. 

This deviation from protocol is considered not to affect the integrity or validity of the study. 
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4. Maintenance of Records

This report has been compiled from original data, which will be stored, together with a copy

of the final report, a copy of the protocol and amendments in Huntingdon Life Sciences’ 

Archives, Huntingdon Life Sciences Ltd, Eye, Suffolk, UK.

Records will be retained for a minimum period of at least one year from the date of issue of 

the final report. At the end of the one year retention period the Sponsor will be contacted and 

advice sought on the future requirements. Under no circumstances will any item be discarded 

without the Sponsor’s knowledge.

Huntingdon Life Sciences will retain the Quality Assurance records relevant to this study for 

twenty years, and a copy of the final report in its archives indefinitely.
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5. Results

5.1 Chemical analysis

The results of chemical analysis are given in Table 1 and example chromatograms are given 

in Appendix 3. 

At all concentrations, the intended exposure levels of were achieved between 70 

to 98% of their nominal values. The slightly low concentrations are considered to have 

resulted from a slightly lower solubility at the lower temperature for an acute fish study when 

compared with the physiochemical properties study (20oC with approx. 15oC for this fish 

study). The concentrations were maintained during the test (between 79 to 136% of their 

starting values). Based on a geometric mean, overall mean measured levels were 0.950, 2.19, 

4.65, 10.5 and 24.5 mg/L and these values have been used in the determination of study endpoints.

5.2 Mortality and observations

No mortality occurred during the 96 hour test, therefore the 96 hour LC50 could not be 

calculated but must be greater than 24.5 mg/L.

A chronological record of treatment-related effects is given in Table 2. One fish was affected 

at 0.950, 4.65, 10.5 and 24.5 mg/L during most of the test; additionally at 24.5 mg/L, three 

fish were affected at 24 hours only. Treatment-related effects comprised hyperventilation, 

loss of body colour, loss of co-ordination and spiral swimming.

The ‘no-observed effect concentrations’ (NOEC) was 10.5 mg/L and the ‘lowest observed 

affect concentration’ (LOEC) was 24.5 mg/L.

5.3 Environmental parameters

The measurements of water quality (temperature, pH, concentrations of dissolved oxygen and 

total hardness) are summarised in Table 3; they remained within acceptable limits throughout 

the study with the exception of total hardness before commencement of the test (see 

deviations from Protocol section). 

At all concentrations, the test media were colourless.
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6. Conclusions

The acute toxicity of to rainbow trout (Oncorhynchus mykiss) has been assessed, 

under semi-static conditions.

No mortality occurred during the 96 hour test; therefore, the 96 hour LC50 could not be 

calculated but was considered under the conditions of this study to be greater than 24.5 mg/L.

The 96 hour ‘no-observed effect concentration’ (NOEC) is considered to be 10.6 mg/L.
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Table 1 Measured concentrations

Nominal

concentrations

(mg/L)

Measured concentrations Overall mean*

(mg/L) 0 hours (F) 24 hours (E) % t0

72 hours (F) 96 hours (E) % t72 mg/L %N mg/L %N mg/L %N mg/L %N

Control nd - nd - - nd - nd - - nd

1.17 0.956 82 0.962 82 101 0.850 73 1.04 89 122 0.950

2.58 2.16 84 2.16 84 100 2.11 82 2.33 90 110 2.19

5.68 4.93 87 4.44 78 90 4.42 78 4.82 85 109 4.65

12.5 12.1 97 9.61 77 79 8.77 70 11.9 95 136 10.5

27.5 27.0 98 23.7 86 88 21.2 77 26.6 97 125 24.5

nd None detected (less than the limit of detection = 0.04 mg/L; Appendix 3)

% ti Measured concentration after 24 or 96 hours expressed as a percentage of the starting concentration (0 and 72 hours)

F Freshly prepared medium

E Expired medium

%N Measured concentration expressed as a percentage of the nominal concentration (calculated using unrounded value)

* Geometric mean

- No measurement required
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Table 2 Sub-lethal effects

concentrations

(mg/L) Abnormality

Initial population = 7 fish / concentration

Nominal Measured 2 h 4 h 24 h 48 h 72 h 96 h

Control nd No abnormalities detected 7/7 7/7 7/7 7/7 7/7 7/7

1.17 0.950 No abnormalities detected 6/7 5/7 6/7 6/7 6/7 6/7

Hyperventilation 0/7 1/7 0/7 0/7 0/7 0/7

2.58 2.19 No abnormalities detected 7/7 7/7 7/7 7/7 7/7 7/7

5.68 4.65 No abnormalities detected 7/7 6/7 6/7 6/7 6/7 6/7

Darkened pigmentation 0/7 1/7 1/7 1/7 1/7 1/7

12.5 10.5

No abnormalities detected 7/7 5/7 6/7 5/7 5/7 5/7

Darkened pigmentation 0/7 1/7 0/7 0/7 0/7 0/7

Curved spine 0/7 1/7 1/7 1/7 1/7 1/7

27.5 24.5

No abnormalities detected 6/7 4/7 3/7 4/7 4/7 4/7

Darkened pigmentation 0/7 1/7 1/7 0/7 0/7 0/7

Loss of body colour 0/7 1/7 1/7 1/7 1/7 1/7

Loss of co-ordination 0/7 0/7 3/7 0/7 0/7 0/7

Spiral swimming 0/7 0/7 3/7 0/7 0/7 0/7

x/y Number of fish affected / number of fish surviving

nd None detected (less than the limit of detection = 0.04 mg/L; Appendix 3)
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Table 3 Environmental parameters

concentrations (mg/L)

TemperatureoC

pH Dissolved oxygen 

% ASV

Total 

hardness 

(mg/L)*

Nominal Measured Min Max Min Max Min Max 0 hours

Control nd 14.5 15.7 7.65 8.05 93 101 161

1.17 0.952 14.1 15.7 7.72 8.17 94 102 -

2.58 2.19 14.1 15.7 7.79 8.25 95 101 -

5.68 4.65 14.2 15.7 7.78 8.26 94 101 -

12.5 10.6 14.5 15.7 7.80 8.27 94 100 -

27.5 24.6 14.8 15.8 8.01 8.26 94 101 163

ASV Air saturation value (100% ASV = 9.09 mgO2/L at 20oC).

* As CaCO3

nd None detected (less than the limit of detection = 0.04 mg/L; Appendix 3)

Continuous monitoring of vessel media temperature = 14.1 to 16.0°C 
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Appendix 1 Certificate of analysis

Awaiting supply from sponsor
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Appendix 2 Typical water quality characteristics of the diluent supply

Diluent water comprised filtered dechlorinated tap water blended with tap water that had 

been softened and subsequently treated by reverse osmosis to a hardness of 150-200 mg/L as 

CaCO3. Analysis of a sample taken in January 2013 gave the following results:

Colony count 37°C 0 / 1 mL

Coliform bacteria/ 100mL 2 / 100 mL

E. coli 0 / 100 mL

μg/L

Organochlorine pesticides <1.24

Organophosphorus pesticides <3.30

Polychlorinated biphenyls <0.007

mg/L

Aluminium <0.0200

Ammonium (as NH4) <0.01

Arsenic <0.0020

Boron 0.0296

Cadmium 0.00031

Calcium 51

Carbon (total organic) 0.55

Chloride 18.7

Residual disinfectant (total chlorine) <0.02

Residual disinfectant (free chlorine) <0.02

Chromium <0.0020

C.O.D. 3.62

Cobalt <0.0020

Copper 0.0248

Fluoride <0.05

Iron <0.015

Lead 0.0006

Magnesium 3.010

Mercury 0.00008

Nitrate (as NO3) 4.66

Nitrite (as NO2) <0.05

Nickel 0.00309

Phosphorus <0.10

Potassium 1.650

Silver 0.00180

Sodium 8.360

Tin <0.002

Suspended solids <2.0

Zinc 0.0198

Conductivity (20°C) 341 μS/cm

Turbidity <2.0 NTU

NTU Nephelometric turbidity units.

No claim of compliance is made for the above data.
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Appendix 3 The determination of in dechlorinated tap water

1. Method

1.1 General principle

Test solutions were diluted with water:acetonitrile:formic acid (95:5:0.1 v:v:v) prior to 

quantitation by liquid chromatography with tandem mass spectrometric detection system

(LC-MS/MS).

Materials Grade

Actonitrile HPLC

Ammonium formate AR

Formic acid AR

Methanol HPLC

Water HPLC

1.1.1 Test article fortifying and calibration solutions

Some standard solutions used in this study have been shared with other GLP studies being 

performed for the same sponsor. This is considered to have no impact on the integrity or 

validity of this study.

A weighed amount of was dissolved in acetonitrile to produce a 1 mg/mL stock 

standard solution. The stock standard was diluted progressively with methanol to give 

fortification standard solutions. The appropriate fortification standard solution was further 

diluted with water:acetonitrile:formic acid (95:5:0.1 v:v:v) to produce a series of calibration 

solutions in the range 0.1 to 5 ng/mL.

1.1.2 Typical preparation of reagents

Preparation of water:acetonitrile:formic acid (95:5:0.1 v:v:v):

Acetonitrile (50 mL) and formic acid (1 mL) were thoroughly mixed with water (950 mL) 

prior to use.

Preparation of water:methanol:formic acid (90:10:0.1 v:v:v) + 0.01M ammonium formate:

Water (900 mL) was thoroughly mixed with methanol (100 mL), formic acid (1 mL) and 

ammonium formate (0.63 g) prior to use.

Preparation of 0.1% formic acid in methanol:

Methanol (1000 mL) was thoroughly mixed with formic acid (1 mL) prior to use.

NOTE – to prepare different volumes of the above reagents, the ratio of the individual 

components was maintained.
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1.2 Procedure

An aliquot (10 mL) of the sample was taken and fortified if required. 

Water:acetonitrile:formic acid (95:5:0.1 v:v:v) (10 mL) was added and mixed well. The 

sample was diluted by a factor of 200 with water:acetonitrile:formic acid (95:5:0.1 v:v:v). 

Any further dilutions, where required, were performed with water:acetonitrile:formic acid 

(95:5:0.1 v:v:v) prior to quantitation by LC-MS/MS.

1.2.1 Instrumentation

LC-MS/MS conditions

Instrument: AB Sciex API 4000

Data system: Analyst v.1.4.2

Ionisation mode: Ionspray positive

Ion monitoring details: MRM m/z 186>57

Column: Phenomenex aqua C18 (5 cm  2 mm)

Mobile phase A: Water:methanol:formic acid (90:10:0.1 v:v:v) + 0.01M 

ammonium formate

Mobile phase B: Methanol + 0.1% formic acid (by volume)

Pump programme: 60:40 A:B isocratic

Cycle time: 8 minutes

Flow rate: 0.3 mL/min

Injection volume: 20 μL

Retention time: Approximately 1.4 minutes

LOQ: 0.1 mg/L

LOD: 0.1 ng/mL (equivalent to 0.04 mg/L in aqueous media)
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1.2.2 Calculation of results

Test samples were quantified using the following equation:

Residue found (mg/L) = 

1000

1

 x D x 

M

1

x x 

Where x (residue concentration in final solution) was calculated using the linear regression:

y = m x + c where x (concentration in ng/mL) = 

y  c

m

c = intercept

m = slope

y = peak area of sample

M = matrix concentration (mL/mL)

D = dilution factor
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2.0 Results and Discussion

2.1 Calibration

The chromatographic response to was shown to be linear over the range of 

concentrations 0.1 to 5 ng/mL. Typical calibration data is presented in Table 1 and Figure 1. 

Typical chromatograms of calibration standards are presented in Figures 2 and 3.

2.2 Validation

The methodology for the determination of residues in dechlorinated tap water was 

validated at 0.1 mg/L and 30 mg/L. The validation data are presented in Table 2 and 

summarised in the following table:

Fortification level

(mg/L) Mean recovery (%) CV (%)

0.1 96 1.4

30 94 4.9

(95) (3.5)

( ) Overall values

2.3 Limit of quantitation (LOQ)

The limit of quantitation is defined as the lowest fortification level at which acceptable 

recovery data are obtained. The validation of the methodology for the determination of 

residues of in dechlorinated tap water, demonstrated that it can be accurately 

determined at a limit of quantitation (LOQ) of 0.1 mg/L.

2.4 Limit of detection (LOD)

The limit of detection of the method is defined as the value of the lowest calibration standard 

chromatographed that gave a peak height to baseline noise ratio 3. For this study the limit 

of detection was 0.1 ng/mL (equivalent to 0.04 mg/L in dechlorinated tap water).

2.5 Samples

Samples of dechlorinated tap water from the test were analysed in suitably sized batches 

along with control samples fortified with which acted as procedural recovery

samples. Procedural recoveries performed on control dechlorinated tap water were in the 

range 70 to 110% confirming the validity of the methodology on each day of analysis. 

Typical chromatograms of untreated, untreated fortified with and treated samples

are presented in Figures 4 to 7.
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Table 1 Typical standard calibration for by LC-MS/MS

Standard concentration (ng/mL) Peak area

0 0

0.1 1.09054e+004

0.2 1.65429e+004

0.4 3.34644e+004

0.8 6.64350e+004

1 7.38713e+004

2 1.51181e+005

3 2.46303e+005

4 3.13915e+005

5 3.93727e+005

Calibration curve: y = 78773.8 x + 657.708 (batch 4)

r = 0.9995

Where x = concentration

y = peak area

r = regression coefficient
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Table 2 Validation of in dechlorinated tap water by LC-MS/MS

Fortification 

level Replicate Analyte Detected Recovery Mean Standard Deviation

Relative 

Standard 

Deviation

Unit: mg/L Unit: mg/L % % % %

Control A ND -

Control B ND -

0.1 A 0.0972 97

0.1 B 0.0941 94 96 1.3 1.4

0.1 C 0.0958 96

0.1 D 0.0945 95

0.1 E 0.0973 97

30 A 29.1 97

30 B 28.6 95 94 4.6 4.9

30 C 26.3 88

30 D 27.5 92

30 E 29.9 100

Overall mean (%) 95

Overall standard deviation (%) 3.3

Overall relative standard deviation (%) 3.5

General Notes:

ND – Not detected (<LOD of 0.04 mg/L)

Control aquatic media sample used – 13/00/A3234

Data system: Analyst v.1.4.2
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Figure 1 Typical standard chemical calibration for by 

LC-MS/MS

CEF0010_B4.rdb : "Linear" Regression ("No" weighting): y = 78773.8 x + 657.708 (r = 0.9994752)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0

Concentration, ng/mL

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

Area, counts
Huntingdon Life Sciences: CEF0010

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Figure 2 Typical chromatogram of a 0.1 ng/mL calibration 

standard

Sample Name: "Cal 0.1 ng/mL" Sample ID: "" File: "CEF0010_B414.wiff"

Peak Name: " Mass(es): "186.0/57.0 amu"

Comment: "" Annotation: ""

Sample Index: 1 

Sample Type: Standard 

Concentration: 0.100 ng/mL 

Calculated Conc: 0.130 ng/mL 

Acq. Date: 19/08/2013 

Acq. Time: 17:43:14 

Modified: No 

Proc. Algorithm: IntelliQuan - IQA 

Min. Peak Height:1000.00 cps

Min. Peak Width: 0.00 sec

Smoothing Width: 9 points

RT Window: 6.00 sec

Expected RT: 1.41 min

Use Relative RT: No 

Int. Type: Valley 

Retention Time: 1.43 min

Area: 1.09054e+004 counts

Height: 1.68e+003 cps

Start Time: 1.33 min

End Time: 1.63 min

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5

Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

4.4e4

4.6e4

4.8e4

5.0e4

5.2e4

5.4e4

5.6e4

5.8e4

Intensity, cps

1.431.48

0.14 0.38 0.70 0.91 1.16 1.37 1.66 2.04 2.27 2.41 2.83 3.07 3.21 3.61 3.86 4.00 4.44 4.55 4.81 5.04 5.24 5.43 5.68 5.87 5.99 6.47 6.54 6.876.92 7.45 7.12 7.607.77

Figure 3 Typical chromatogram of a 5 ng/mL calibration 

standard

Sample Name: "Cal 5 ng/mL" Sample ID: "" File: "CEF0010_B420.wiff"

Peak Name: " Mass(es): "186.0/57.0 amu"

Comment: "" Annotation: ""

Sample Index: 1 

Sample Type: Standard 

Concentration: 5.00 ng/mL 

Calculated Conc: 4.99 ng/mL 

Acq. Date: 19/08/2013 

Acq. Time: 18:35:16 

Modified: No 

Proc. Algorithm: IntelliQuan - IQA 

Min. Peak Height:1000.00 cps

Min. Peak Width: 0.00 sec

Smoothing Width: 9 points

RT Window: 6.00 sec

Expected RT: 1.41 min

Use Relative RT: No 

Int. Type: Valley 

Retention Time: 1.44 min

Area: 3.93727e+005 counts

Height: 5.82e+004 cps

Start Time: 1.26 min

End Time: 1.85 min

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5

Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

4.4e4

4.6e4

4.8e4

5.0e4

5.2e4

5.4e4

5.6e4

5.8e4

Intensity, cps

1.44

1.66

0.13 0.40 0.83 1.25 2.14 2.57 2.80 2.88 3.91 4.34 4.43 4.66 5.005.29 5.62 5.69 5.88 6.21 6.80 7.12 7.67 7.89 0.75 3.31 6.61
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Figure 4 Chromatogram of an untreated dechlorinated tap water sample 

(analytical identification 13/00/A3281 Con)

Sample Name: "A3281 Con" Sample ID: "" File: "CEF0010_B419.wiff"

Peak Name: " Mass(es): "186.0/57.0 amu"

Comment: "" Annotation: ""

Sample Index: 1 

Sample Type: Unknown 

Concentration: N/A 

Calculated Conc: 0.00 ng/mL 

Acq. Date: 19/08/2013 

Acq. Time: 18:26:35 

Modified: No 

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5

Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

4.4e4

4.6e4

4.8e4

5.0e4

5.2e4

5.4e4

5.6e4

5.8e4

Intensity, cps

0.38 1.44 0.31 0.430.83 0.99 1.38 1.51 1.77 2.17 2.412.57 2.77 3.04 3.273.43 3.79 3.88 4.06 4.44 4.574.614.914.95 5.27 5.605.66 5.875.916.31 6.41 6.52 6.88 7.11 7.42 7.74 7.80

Figure 5 Chromatogram of an untreated dechlorinated tap water sample 

fortified with at 10 mg/L (analytical identification 

13/00/A3281 F10)

Sample Name: "A3281 F10" Sample ID: "" File: "CEF0010_B421.wiff"

Peak Name: " Mass(es): "186.0/57.0 amu"

Comment: "" Annotation: ""

Sample Index: 1 

Sample Type: Unknown 

Concentration: N/A 

Calculated Conc: 26.8 ng/mL 

Acq. Date: 19/08/2013 

Acq. Time: 18:43:57 

Modified: No 

Proc. Algorithm: IntelliQuan - IQA 

Min. Peak Height:1000.00 cps

Min. Peak Width: 0.00 sec

Smoothing Width: 9 points

RT Window: 6.00 sec

Expected RT: 1.41 min

Use Relative RT: No 

Int. Type: Valley 

Retention Time: 1.44 min

Area: 2.11570e+005 counts

Height: 3.08e+004 cps

Start Time: 1.27 min

End Time: 1.78 min

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5

Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

4.4e4

4.6e4

4.8e4

5.0e4

5.2e4

5.4e4

5.6e4

5.8e4

Intensity, cps

1.44
Huntingdon Life Sciences: CEF0010

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Figure 6 Chromatogram of a test sample at 12.5 mg/L of on 

Day 0 (analytical identification 13/CEF/A3295)

Sample Name: "A3295" Sample ID: "" File: "CEF0010_B425.wiff"

Peak Name: " Mass(es): "186.0/57.0 amu"

Comment: "" Annotation: ""

Sample Index: 1 

Sample Type: Unknown 

Concentration: N/A 

Calculated Conc: 30.1 ng/mL 

Acq. Date: 19/08/2013 

Acq. Time: 19:18:36 

Modified: Yes 

Proc. Algorithm: IntelliQuan - IQA 

Min. Peak Height:1000.00 cps

Min. Peak Width: 0.00 sec

Smoothing Width: 9 points

RT Window: 6.00 sec

Expected RT: 1.41 min

Use Relative RT: No 

Int. Type: Manual 

Retention Time: 1.44 min

Area: 2.37925e+005 counts

Height: 3.31e+004 cps

Start Time: 1.26 min

End Time: 1.92 min

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5

Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

4.4e4

4.6e4

4.8e4

5.0e4

5.2e4

5.4e4

5.6e4

5.8e4

Intensity, cps

1.44

Figure 7 Chromatogram of a test sample at 12.5 mg/L of on 

Day 1 (analytical identification 13/CEF/A3352)

Sample Name: "A3352" Sample ID: "" File: "CEF0010_B524.wiff"

Peak Name: " Mass(es): "186.0/57.0 amu"

Comment: "" Annotation: ""

Sample Index: 1 

Sample Type: Unknown 

Concentration: N/A 

Calculated Conc: 24.0 ng/mL 

Acq. Date: 20/08/2013 

Acq. Time: 18:57:57 

Modified: No 

Proc. Algorithm: IntelliQuan - IQA 

Min. Peak Height:1000.00 cps

Min. Peak Width: 0.00 sec

Smoothing Width: 9 points

RT Window: 6.00 sec

Expected RT: 1.41 min

Use Relative RT: No 

Int. Type: Valley 

Retention Time: 1.44 min

Area: 1.72790e+005 counts

Height: 2.69e+004 cps

Start Time: 1.29 min

End Time: 1.83 min

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5

Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

4.4e4

4.6e4

4.8e4

5.0e4

5.2e4

5.4e4

Intensity, cps

1.44

1.64

0.05 0.43 0.75 1.06 1.32 1.88 2.32 2.44 2.91 3.04 3.48 3.67 3.79 4.17 4.43 4.54 4.84 5.12 5.25 5.92 6.12 6.36 6.59 6.67 6.99 7.48 7.55 7.79
Huntingdon Life Sciences: CEF0010

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Appendix 4 Compliance statement