Wastewater pollution undermines coastal marine protection: Implications for 30x30 and effective conservation

David E. Carrasco Rivera  & Amelia S. Wenger 

Articles and information - Environment Management, Ecology, Conservation, Biodiversity, Aquaculture

PCR Technique for Pathogens Detection in Aquaculture

By Dr Leonardo Galli (Associate Director, Aquaculture)

I began performing PCR (polymerase chain reaction) analyses in 1999, while working for a major shrimp (prawn) company in Ecuador. Since then, I implemented and operated diagnostic laboratories for aquaculture companies in Ecuador, Brazil, Mozambique and Thailand.

The global outbreak of White Spot Virus in shrimp farming in the late 1990’s popularised the application of the PCR technique for the screening of brood-stock and post-larvae before stocking. Currently this technique is widely used in diseases control and health surveillance programs in shrimp and fish farming.

One of the limitations that farmers occasionally encountered was contradictory results; similar samples sent to different laboratories yielded different results. 

The PCR is the central analytical step within a broader workflow. To understand the constraints affecting test performance, one must consider pre-analytical (upstream), analytical and post-analytical (downstream) steps. 

This article will be limited to addressing the limitations of the pre-analytical phase of the process, considering that this may help aquaculture producers understand the practical implications in the PCR technique.

Upstream Constraints 

The upstream phase encompasses everything from sample collection to the introduction of purified nucleic acid into the PCR master mix. Constraints here are physical and chemical in nature.

Sample Collection

The way in which a sample is collected depends on the objectives of the sampling. 

If there is a tank or pond with animals showing signs of disease or erratic behaviour, a directed (targeted) sample can be carried out, by selecting these animals to conduct the test. The number of individuals in the sample is not important, between five to ten will be sufficient, as long as they all show signs of disease. 

If the objective of the sampling is to conduct a screening or health surveillance in an apparently healthy population, the size of the sample is important. The first assumption is that the individuals in the population are randomly distributed. The sample size will be affected by the population size and level of prevalence of the pathogen in that population. The lower the prevalence, the larger the sample size that must be collected. This has profound economic repercussions, due to the cost of the analysis. If the population is over 100.000 and the selected prevalence is 2% the sample size should be 165 individuals. If the selected prevalence is 10%, the sample size go down to 34 individuals. Increasing the prevalence value implies assuming the risk that the PCR test will not be positive if the pathogen is below that limit. 

Another procedure to reduce the number of tests to be performed is by pooling the samples. This is a valid technique, but the risk lies in the dilution effect of the pathogen in the mixed sample. The size of the pool determines the number of tests to perform. For a large population 34 pools of 5 individual each will be needed to detect a pathogen with a prevalence of 2%, or 17 pools of 10 individuals each for the same prevalence. The risk is to go under the limit of detection of the test and get a false negative. 

This is known as a “management risk decision” and, in many cases, the selected prevalence level depends on the severity of the pathogen in question. There are numerous tools on the internet to estimate sample size based on population size.   

Depending on the pathogen, the organs or tissues to be sampled are important. Some pathogens have specific tissues tropism. The result will be negative if the wrong organ is used in the analysis. 

Sample Fixation

This is a critical step that can affect the test result.

The most common fixative is 95% Ethyl Alcohol. The samples should be immersed in alcohol at a ratio of 1:10 (1 volume of sample per 10 volumes of alcohol). For long-term storage in RNA virus testing, other special fixatives can be used. Frozen samples can also be used if they are frozen immediately after collection.

Special care should be taken avoiding contamination with PCR inhibitors such as calcium carbonate (which is commonly used in disposable gloves). 

Nucleic Acid Extraction 

Nucleic acid extraction involves cell lysis, purification and in some methods, elution. It can be performed in solid phase (spin columns), Phenol-Chloroform extraction, lysis buffer salt extraction, magnetic beads, etc.   

Co-purified substances (haemoglobin, heparin, bile salts, etc.) can inhibit the PCR reaction. Extreme care must be taken to avoid these types of compounds in the samples.

It is recommended to include house-keeping gene amplification primers (as internal control) to ensure that the premix reagents work properly.

The excess of target nucleic acid can saturate the reagents or interfere with probe binding, paradoxically leading to a false negative or an underestimation of concentration. This is a critical upstream constraint in quantitative PCR (qPCR).

Environmental impacts of oil spills and fires

By Gino Sabatini, Associate Director

19 March 2026 

I was the Marine & Coastal Manager overseeing a study to ascertain the adverse long-term impacts of the oil spills on sensitive environmental receptors, including coastal and marine habitats, fisheries and coral reefs. The result of our surveys showed that oil was still present in the sediment all the way from Damman (Saudi Arabia) to areas beyond the Kuwait border. Oil was present on all shoreline types: sandy beaches, rocky beaches, tidal mudflats, mangroves and salt marshes. Even liquid oil was still present, especially in mangrove, salt marsh, mudflat, and some fine-grained sand beaches as shown in the following photo:



On rocky beaches the oil had dried to a consistency equivalent to asphalt (tar mats) as shown below:



Dried oil on rocks - appearing a tar mat on a beach north of Abu Ali island. Photo by Gino Sabatini, 2002.



However, most of the oiled sediment was buried several centimeters deep between clean sediment layers as shown below: 



From Salem, M. & A. Hamid (2016) Stabilisation/solidification (S/S) technique and its application in Saudi Arabia. International Journal of Environment and Sustainability, vol 5, No. 1, pp 45-60.

The oil formed a continuous layer traversing all intertidal habitats regardless of biotopes: sand beach, tidal flat, salt marsh, or mangrove.  The Saudi Arabian shoreline, from north of Damman to the Kuwait border was still contaminated with an average thickness of the oil layer of approximately 10cm, amounting to a volume of 9 million cubic meters. 

These surveys revealed that 64% of mangroves, 70% of salt marshes, 88% of tidal flats, 90% of sand beaches and 90% of rocky shores remained impacted with lower diversity than that found in non-oil polluted control shores.  As a result, predicted recovery times were revised and extended to over a century for habitats such as salt marshes.[1]



In 2005  the UNCC awarded Saudi Arabia 1.1billion USD in for shoreline remediation works and other environmental damage claims, of which half was solely for shoreline environmental damages.  Other countries and individuals who lodged claims were also compensated.  It was the first time the United Nations had awarded monetary compensation related to claims for environmental damages as a result of war.  (https://uncc.un.org/en). The ongoing war in the Arabian Gulf may also have severe adverse impacts on the environment. Hopefully, the lessons learned on ecological restoration will be useful for the forthcoming recovery. I shall refer to these in some detail in my next article.

[1]  Jones, DA. Hayes, M. Krupp, F. Sabatini, G. Watt, I. & Weishar, L. 2008  IN: Protecting the Gulf’s Marine Ecosystems from Pollution.  Edited by A.H. Abuzinada, H.-J. Barth, F. Krupp, B. Böer and T.Z. Al Abdessalaam

© 2008 Birkhäuser Verlag/Switzerland

Protecting habitats and biodiversity through effective impact avoidance

Current best-practice guidance on environmental management and impact assessment, such as CIEEM, (2016) and The Equator Principles is based on the ‘mitigation hierarchy’ – avoidance, mitigation and compensation of significant adverse impacts, followed when possible by ‘enhancement’. Avoidance of impacts is the preferred option to protect ecosystems, biological communities and biodiversity. In our previous article, we discussed some technical aspects of coral relocation, which corresponds to the third echelon of the mitigation hierarchy - compensation. This may be followed with specific measures or actions to increase the extent of habitats or biological diversity. 

Impact avoidance is the most effective and less costly way to protect the natural environment, as mitigation and compensation may not entirely deliver the expected results, and are often extremely expensive. It consists in making options that will not harm ecological features – ecosystems, habitats, species, especially those that are rare, sensitive or have been already affected by previous human actions or development. Impact avoidance must be considered at the earliest stage of any project, during its inception and concept design. This approach should be given the highest priority, as it will not only avoid lengthy and expensive studies and the implementation of costly measures for mitigation and compensation. It will also open opportunities for innovative design and nature-based solutions, which can be achieved with the participation of experienced and qualified environmental scientists during these early stages. A common error is to underestimate the potential adverse impacts of the construction and/or operation of a project during the initial design stages. This invariably leads to considerable delay and huge expenses during the later stages. We know about significant infrastructure projects that have been delayed for more than 12 years precisely for this mistake, compounded by unrealistic impact assessments not properly supported by evidence. Most members of EnvCR’s team have been involved in the later remediation of these shortfalls, after the developers realised that they had missed the window of opportunity to incorporate impact avoidance measures to their design. It is a common and expensive mistake. The best way to avoid this error is to seek expert professional advice during these early design stages, from qualified environmental scientists with the necessary level of experience. Failure to do this invariably results in years of delay, large bills and often, in adverse environmental effects as well. We shall discuss the mitigation and compensation measures in our next article.

Coral reefs: Conservation, protection, impact avoidance and mitigation.

Step 2 - Prepare Recipient Site 

Depending on size, substrate is often necessary to re-attach corals.  The type of substrate will often depend upon the size of coral to be relocated.  In the Arabian/Persian Gulf and the Red Sea, for example, coral heads are rarely larger than 1m in width, therefore these are detached and placed in baskets or attached to balloons to be moved to the recipient site (see photos below).

Substrate choice can be as simple as rebar; the same rebar as used for concrete. Other hard materials include concrete or rock.

Step 3- Detachment of Corals

Corals are detached using hammer and chisel, dependant upon coral head size, and placed in baskets for transportation to the recipient site.

Step 4 - Transfer of Corals

The transfer of corals is done underwater, or they can be placed on boats in water containers to be transported to the recipient site.  The amount of coral transported at any one time is dependant upon level of effort - area, or number of coral heads to be relocated). Cranes and buoys can also be used for transporting large coral heads.

Step 5 – Coral attachment

Coral re-attachment should mimic the natural conditions of the coral reef community from which they were removed, including soft corals. Sound understanding of coral reef ecology is essential to ensure that they are placed in assemblages with species they tolerate so as to minimise coral competition and stress.

Step 6 - Cleaning and Maintenance

Areas around the relocated corals will need to be cleaned regularly to avoid overgrowth by algae, especially for the first few weeks following re-attachment.  Frequency of cleaning will depend upon the time of year (during warmer months algal growth is generally increases), natural sedimentation rates, and nearby construction or dredging activities (if occurring).

Step 7 – Monitoring

This is important to ascertain the success of the coral relocation efforts.  The following parameters, at very least, should be monitored: 

●         Coral survivorship (%)

●         Coral Health (disease and bleaching occurrences)